Tnfrsf-binding agents and uses thereof

ABSTRACT

Polypeptides and agents that bind a TNF receptor superfamily protein are disclosed, particularly agents that specifically bind GITR, OX40, or CD40. The polypeptides or agents may include fusion polypeptides, particularly polypeptides comprising GITRL, OX40L, or CD40L and/or bispecific agents. Also disclosed are methods of using the polypeptides or agents for inducing and/or enhancing the immune response, as well as methods for the treatment of diseases such as cancer.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the priority benefit of U.S. Provisional Application No. 62/111,404, filed Feb. 3, 2015, U.S. Provisional Application No. 62/153,272, filed Apr. 27, 2015, U.S. Provisional Application No. 62/154,008, filed Apr. 28, 2015, U.S. Provisional Application No. 62/218,956, filed Sep. 15, 2015, and U.S. Provisional Application No. 62/259,129, filed Nov. 24, 2015 each of which is hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention generally relates to agents that bind tumor necrosis factor receptor superfamily members, particularly agents comprising the extracellular domain of GITRL, OX40L, or CD40L. The invention also relates to methods of using the agents for the modulation of immune responses and/or the treatment of diseases such as cancer.

BACKGROUND OF THE INVENTION

The basis for immunotherapy is the manipulation and/or modulation of the immune system, including both innate immune responses and adaptive immune responses. The general aim of immunotherapy is to treat diseases by controlling the immune response to a “foreign agent”, for example a pathogen or a tumor cell. However, in some instances immunotherapy is used to treat autoimmune diseases which may arise from an abnormal immune response against proteins, molecules, and/or tissues normally present in the body. Immunotherapy may include methods to induce or enhance specific immune responses or to inhibit or reduce specific immune responses.

The immune system is a highly complex system made up of a great number of cell types, including but not limited to, T-cells, B-cells, natural killer cells, antigen-presenting cells, dendritic cells, monocytes, and macrophages. These cells possess complex and subtle systems for controlling their interactions and responses. The cells utilize both activating and inhibitory mechanisms and feedback loops to keep responses in check and not allow negative consequences of an uncontrolled immune response (e.g., autoimmune diseases or a cytokine storm).

The concept of cancer immunosurveillance is based on the theory that the immune system can recognize tumor cells, mount an immune response, and suppress the development and/or progression of a tumor. However, it is clear that many cancerous cells have developed mechanisms to evade the immune system which can allow for uninhibited growth of tumors. Cancer/tumor immunotherapy (immunooncology) focuses on the development of new and novel agents that can activate and/or boost the immune system to achieve a more effective attack against tumor cells resulting in increased killing of tumor cells and/or inhibition of tumor growth.

BRIEF SUMMARY OF THE INVENTION

Proteins belonging to the tumor necrosis factor receptor superfamily (TNFRSF) and their ligands (TNFSF) are intimately involved in the activation, differentiation, and survival of cells of the immune system. TNFRSF members include, but may not be limited to, 4-1BB, BAFF, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL R1, DcTRAIL R2, DR3, DR6, EDA2R, EDAR, Fas (CD95), GITR, HVEM, lymphotoxin beta R, NGFR, osteoprotegerin, OX40, RANK, RELT, TACI, TNFRH3, TNF R1, TNF R2, TRAIL R1, TRAIL R2, TRAIL R3, TRAIL R4, TROY, and TWEAK R. Receptors for TNF family ligands are oligomeric, type I or type III transmembrane proteins that contain multiple extracellular cysteine-rich domains. Several of these receptors also contain intracellular death domains (DDs) that recruit caspase-interacting proteins following ligand binding to initiate the extrinsic pathway of caspase activation. Other TNF superfamily receptors that lack death domains bind TNF receptor-associated factors and activate intracellular signaling pathways that can lead to proliferation or differentiation. These receptors can also initiate apoptosis, but they do so via indirect mechanisms. In addition to regulating apoptosis, several TNF superfamily receptors are involved in regulating immune cell functions such as B-cell homeostasis and activation, natural killer cell activation, and T-cell co-stimulation. Several others regulate cell type-specific responses such as hair follicle development and osteoclast development.

TNFSF members include, but may not be limited to, 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30 ligand, CD40 ligand (CD40L), EDA, EDA-A1, EDA-A2, Fas ligand (CD95L), GITR ligand (GITRL), LIGHT, lymphotoxin, lymphotoxin beta, lymphotoxin-alpha, OX40 ligand (OX40L), TL1A, TNF-alpha, TRAIL, TRANCE, and TWEAK. Most TNF ligands are type II transmembrane proteins whose extracellular domains can be cleaved by specific metalloproteinases to generate soluble cytokines. Cleaved and non-cleaved ligands are active as non-covalent homotrimers except for lymphotoxin beta (which forms heterotrimers with TNF-beta) and BAFF (which forms heterotrimers with APRIL). TNF family ligands are characterized by a stalk of varying length connecting the transmembrane domain to the core region, which contains the hallmark structure of TNF family ligands, the TNF homology domain (THD) or TNF domain. The TNF domain is an anti-parallel beta-pleated sheet sandwich with a “jelly-roll” topology. Conserved residues within the beta-strands provide specific inter-subunit contacts, which stabilize the trimeric structure. Sequences in the loops connecting adjacent beta-strands are family member-specific and are important for conferring receptor specificity. Interestingly, GITRL (glucocorticoid-induced TNF-related ligand; TNFSF18) appears to be relatively loosely associated as a trimer as compared to other TNF family members, and has been shown to also exist in dimeric states. Further, there is evidence that GITRL trimers can themselves associate to form “superclusters” (Zhou et al., 2008, PNAS, 105:5465-5470). Crosslinking of GITRL to stabilize the trimer formation resulted in enhanced activity (Wyzgol et al., 2009, J. Immunol., 183:1851-1861). These results have led to the suggestion that GITRL may exist in a range of oligomeric states ranging from dimers to trimers, to superclusters of trimers, and that these states may result in a range of GITR activity from weak to robust, respectively.

Because agonist antibodies targeting members of the TNFR superfamily are generally dimeric molecules with each arm of the antibody binding one subunit of a TNFR, the inventors hypothesized that they may not be able to fully recapitulate the signaling impact of the native trimeric TNF family member. In light of the data suggesting that GITRL may achieve distinct signaling levels through variation of the oligomerization status of GITRL, it was hypothesized that a therapeutic agent that presents the GITRL in a stable trimeric form could be more active than an agonist GITR antibody at eliciting signaling, and that such a trimeric GITRL form might therefore be a superior immunotherapeutic agent. Further, it was hypothesized that a therapeutic agent that provided two or more GITRL trimers might achieve the impact of a GITRL “supercluster” and be more effective than an agonist GITR antibody. It is believed that this theory would be true for most, if not all, of the TNFR ligands in comparison to TNFR agonist antibodies.

As GITRL has a relatively loose association as a trimer, the means of achieving effective production of a stable therapeutic GITRL trimer is not clear. One strategy to stabilize the GITRL trimer is to express the three subunits of the trimer as a single polypeptide. It has previously been shown that TNF family members can be expressed as a single chain trimer (US Application Publication Nos. 2007/0286843 and 2011/0162095). However, a major drawback of previous single chain TNF family member trimer variants has been the introduction of exogenous linker sequences interconnecting the three subunits of the trimer. Such linkers may introduce potential instability and lability to the trimer, and/or provide a source of potential immunogenicity as the linkers are foreign sequences.

The crystal structure of a human GITRL trimer was examined by the inventors and it was observed that the N-terminal amino acid residues from one monomer and the C-terminal amino acid residues from a second monomer were in close proximity to each other. This suggested that a very short span of amino acid residues, for example only 3-7 residues, might be sufficient to bridge the distance between each monomer and thereby enable a single chain GITRL trimer to be produced without a long peptide linker. Upon further analysis of GITRL it was recognized that there exists a “stalk” of several amino acids between the transmembrane domain and the TNF homology domain of the protein. It was hypothesized that it could be possible to utilize this short stalk region to bridge the distance from the C-terminus of a GITRL monomer to the N-terminus of an adjacent GITRL monomer and in this fashion construct a single chain GITRL trimer that was devoid of exogenous peptide linker sequences.

Other ligands of the TNF superfamily have similar structures, however the size and the amino acid composition of the stalk region varies with each ligand. Furthermore, the native trimer structure of each TNFSF ligand is slightly unique and the amount of the stalk region required for proper folding of each single chain trimeric fusion protein may be different for each TNFSF ligand. For some TNFSF ligands, particularly where the stalk region is very long, only a fragment of the stalk region may be used to generate a single chain trimer.

The present invention provides a variety of polypeptides and agents that bind human tumor necrosis factor receptor superfamily (TNFRSF) members. As used herein, the term “agent” includes, but is not limited to, polypeptides, fusion proteins, homodimeric molecules, and heterodimeric molecules. In some embodiments, a polypeptide or agent binds human glucocorticoid-induced tumor necrosis factor-related protein (GITR). In certain embodiments, the polypeptide or agent is a GITR agonist. In some embodiments, the polypeptide or agent that binds GITR is a soluble GITR ligand (GITRL). In some embodiments, a polypeptide or agent binds human OX40. In certain embodiments, the polypeptide or agent is an OX40 agonist. In some embodiments, the polypeptide or agent that binds OX40 is a soluble OX40 ligand (OX40L). In some embodiments, a polypeptide or agent binds human CD40. In certain embodiments, the polypeptide or agent is a CD40 agonist. In some embodiments, the polypeptide or agent that binds CD40 is a soluble CD40 ligand (CD40L). The invention provides methods of using the polypeptides and agents described herein. In some embodiments, the invention provides methods of using the polypeptides and agents for cancer immunotherapy or immuno-oncology. In some embodiments, the polypeptides and agents are used in methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response. In some embodiments, the polypeptides and agents are used in methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response to cancer, a tumor, and/or tumor cells. In some embodiments, the polypeptides and agents are used in methods of inhibiting the growth of a tumor or tumor cells. In some embodiments, the polypeptides and agents are used in methods for the treatment of cancer. In some embodiments, the methods comprise inhibiting the growth of cancer cells. The invention also provides compositions comprising the agents described herein. In some embodiments, the compositions are pharmaceutical compositions comprising the polypeptides and agents described herein. Polynucleotides encoding the polypeptides and agents and methods of making the agents are also provided.

In one aspect, the invention provides a polypeptide that comprises a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, a polypeptide comprises a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein, wherein at least one of the first, second, or third copies of the extracellular domain or fragment thereof comprises the stalk region of the TNFSF protein. In some embodiments, the the TNFSF protein is selected from the group consisting of: GITRL, OX40L, 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30 ligand, CD40 ligand, EDA, EDA-A1, EDA-A2, Fas ligand, LIGHT, lymphotoxin, lymphotoxin beta, lymphotoxin-alpha, TL1A, TNF-alpha, TRAIL, TRANCE, and TWEAK. In some embodiments, the TNFSF protein is GITRL and any copies of fragments of the extracellular domain are GITR-binding fragments. In certain alternative embodiments, the TNFSF protein is OX40L and any copies of fragments of the extracellular domain are OX40-binding fragments. In certain alternative embodiments, the TNFSF protein is CD40L and any copies of fragments of the extracellular domain are CD40-binding fragments. In some embodiments, at least one, at least two, or all three of the copies of the extracellular domains or fragments thereof comprise the stalk region of the extracellular domain. In some embodiments, the polypeptide further comprises an Fc region. Polypeptides and agents, including without limitation homodimeric agents, heterodimeric agents, and bispecific agents, comprising or consisting of the polypeptides described herein are provided.

In some embodiments, a polypeptide comprises a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein, wherein at least one of the first, second, or third copies of the extracellular domain or fragment thereof comprises the stalk region of the TNFSF protein. In some embodiments, extracellular domain of the TNFSF protein comprises the stalk region or a fragment of the stalk region. In some embodiments, the extracellular domain comprises only a short fragment of the stalk region. In some embodiments, the stalk region is about 4-20 amino acids. In some embodiments, the stalk region is about 4, 5, 6, 7, 8, 9, or 10 amino acids. In some embodiments, the extracellular domain comprises a stalk region (e.g., 4-10 amino acids) directly upstream from the TNF homology domain. In some embodiments, the extracellular domain comprises a stalk region (e.g., 4-10 amino acids) adjacent to the TNF homology domain.

In another aspect, the present invention provides polypeptides and agents that bind GITR (TNFRSF18). In some embodiments, the polypeptide or agent binds human GITR. In some embodiments, the polypeptide or agent binds mouse GITR. In some embodiments, the polypeptide or agent is a soluble protein. In some embodiments, the polypeptide or agent is a soluble protein that binds human GITR. In some embodiments, the polypeptide or agent is a fusion polypeptide. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of GITRL. As used herein, a “copy” of the extracellular domain, or a fragment thereof, of a protein generally refers to a monomer of the extracellular domain of the protein or fragment thereof. For instance, a copy of the extracellular domain, or a fragment thereof, of GITRL generally refers to a monomer of the extracellular domain of the protein, or a fragment thereof, of GITRL. Therefore, multiple “copies” of the extracellular domain generally refers to a dimer (2 copies) of the extracellular domain or a trimer (3 copies) of the extracellular domain of GITRL. In some embodiments, the copies are polypeptides that are exact replicates of the known sequence (e.g., have 100% sequence identity to the native extracellular domain). In certain alternative embodiments, one or more of the copies comprise mutations, such as conservative substitutions, as long as the polypeptide or fusion polypeptide retains the ability to bind to the receptor, i.e., GITR. For example, in certain embodiments, the copies may be polypeptides that have at least about 98%, at least 99%, or 100% sequence identity to the native extracellular domain, or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of human GITRL. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of mouse GITRL. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human GITRL. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a GITR-binding fragment thereof. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human GITRL, wherein at least one of the extracellular domains comprises the “stalk region” of GITRL. In some embodiments, the stalk region of GITRL is LQLETAK (SEQ ID NO:32). In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human GITRL, wherein each extracellular domain comprises the “stalk region” of GITRL. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human GITRL, wherein only the second and third extracellular domains include the “stalk region” of GITRL. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein the polypeptide does not comprise any peptide linkers. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between any of the copies of the extracellular domain or a fragment thereof. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the first copy and the second copy of the extracellular domain of human GITRL or a fragment thereof. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the second copy and the third copy of the extracellular domain of human GITRL or a fragment thereof.

In some embodiments, the agent (e.g., polypeptide) comprises approximately amino acids 71 to 199 of human GITRL. In some embodiments, the polypeptide or agent comprises approximately amino acids 71 to 199 of human GITRL, wherein the sequence of human GITRL is UniProt No. Q9UNG2. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of SEQ ID NO:3 or a fragment thereof. In some embodiments, the agent (e.g., polypeptide) comprises at least two copies of SEQ ID NO:3 or a fragment thereof. In some embodiments, the agent (e.g., polypeptide) comprises three copies of SEQ ID NO:3. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of SEQ ID NO:64. In some embodiments, the agent (e.g., polypeptide) comprises at least two copies of SEQ ID NO:64. In some embodiments, the agent (e.g., polypeptide) comprises three copies of SEQ ID NO:64. In some embodiments, the agent (e.g., polypeptide) comprises SEQ ID NO:5. In some embodiments, the agent (e.g., polypeptide) comprises SEQ ID NO:66. In some embodiments, the agent is a fusion polypeptide or fusion protein. In some embodiments, the fusion protein comprises a non-GITRL polypeptide (i.e., a heterologous protein). In some embodiments, the fusion polypeptide comprises a Fc region. In some embodiments, the non-GITRL polypeptide comprises a Fc region. In some embodiments, the Fc region is from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the non-GITRL polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and light chain form an antigen-binding site. In some embodiments, the non-GITRL polypeptide comprises a single chain antibody or a Fab.

In some embodiments, the agent or polypeptide comprises SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the agent comprises a polypeptide encoded by the hGITRL-hIgG1 plasmid deposited with ATCC and assigned designation number PTA-122112. In some embodiments, the agent or polypeptide comprises SEQ ID NO:8 or SEQ ID NO:9.

In an additional aspect, the invention provides a polypeptide having at least about 90% sequence identity to SEQ ID NO:5 or SEQ ID NO:66, as well as polypeptides or agents comprising such a polypeptide. In some embodiments, the polypeptides or agents comprise a polypeptide consisting of SEQ ID NO:5 or SEQ ID NO:66.

In a further aspect, the present invention provides polypeptides and agents that bind OX40 (TNFRSF4). In some embodiments, the polypeptide or agent binds human OX40. In some embodiments, the polypeptide or agent binds mouse OX40. In some embodiments, the agent is a polypeptide. In some embodiments, the polypeptide or agent is a soluble protein. In some embodiments, the polypeptide or agent is a soluble protein that binds human OX40. In some embodiments, the polypeptide or agent is a fusion polypeptide. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or an OX40-binding fragment thereof, of OX40L (TNFSF4). In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or a fragment thereof, of human OX40L. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of mouse OX40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L, wherein at least one of the extracellular domains comprises the “stalk region” of OX40L. In some embodiments, the stalk region of OX40L is QVSHRYP (SEQ ID NO:55). In some embodiments, the stalk region of OX40L is a variant including but not limited to, ALQVSHRYP (SEQ ID NO:74), SHRYP (SEQ ID NO:75), or HRYP (SEQ ID NO:76). As used herein, the variant stalk regions consist of amino acid sequences of OX40L, i.e., these variant stalk regions do not comprise any exogenous amino acids, such as an exogenous linker. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human OX40L, wherein each extracellular domain comprises the “stalk region” of OX40L or a variant stalk region. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human OX40L, wherein the second and third extracellular domains include the “stalk region” of OX40L or a variant stalk region. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L or an OX40-binding fragment thereof, wherein the polypeptide does not comprise any peptide linkers. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human OX40L or an OX-40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the first copy and the second copy of the extracellular domain of human OX40L or an OX40-binding fragment thereof. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human OX40L or an OX40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the second copy and the third copy of the extracellular domain of human OX40L or an OX40-binding fragment thereof.

In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 50 to 183 of human OX40L. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 51 to 183 of human OX40L. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 53 to 183 of human OX40L. In some embodiments, the polypeptide or agent comprises approximately amino acids 50 to 183, 51 to 183, or 53 to 183 of human OX40L, wherein the sequence of human OX40L is UniProt No. P23510. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:42 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:42 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:42. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:67 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:67 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:67. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:78 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:78 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:78. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:77 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:77 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:77. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:79 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:79 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:79. In some embodiments, the polypeptide or agent comprises SEQ ID NO:44. In some embodiments, the polypeptide or agent comprises SEQ ID NO:69. In some embodiments, the polypeptide or agent comprises SEQ ID NO:70. In some embodiments, the polypeptide or agent comprises SEQ ID NO:71. In some embodiments, the polypeptide or agent comprises SEQ ID NO:72. In some embodiments, the polypeptide or agent is a fusion polypeptide or fusion protein. In some embodiments, the fusion protein comprises a non-OX40L polypeptide (i.e., a heterologous protein). In some embodiments, the fusion polypeptide comprises a Fc region. In some embodiments, the non-OX40L polypeptide comprises a Fc region. In some embodiments, the Fc region is from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the non-OX40L polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and light chain form an antigen-binding site. In some embodiments, the non-OX40L polypeptide comprises a single chain antibody or a Fab.

In some embodiments, the agent or polypeptide comprises SEQ ID NO:45 or SEQ ID NO:46. In some embodiments, the agent or polypeptide comprises SEQ ID NO:47 or SEQ ID NO:48. In some embodiments, the agent or polypeptide comprises SEQ ID NO:80 or SEQ ID NO:81.

In an additional aspect, the invention provides a polypeptide having at least about 90% sequence identity to SEQ ID NO:44, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72, as well as polypeptides or agents comprising such a polypeptide. In some embodiments, the polypeptides or agents comprise a polypeptide consisting of SEQ ID NO:44, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72.

In a further aspect, the present invention provides polypeptides and agents that bind CD40 (TNFRSF5). In some embodiments, the polypeptide or agent binds human CD40. In some embodiments, the polypeptide or agent binds mouse CD40. In some embodiments, the agent is a polypeptide. In some embodiments, the polypeptide or agent is a soluble protein. In some embodiments, the polypeptide or agent is a soluble protein that binds human CD40. In some embodiments, the polypeptide or agent is a fusion polypeptide. In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or a CD40-binding fragment thereof, of CD40L (TNFSF5). In some embodiments, the agent (e.g., polypeptide) comprises at least one copy of the extracellular domain, or a fragment thereof, of human CD40L. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain, or a fragment thereof, of mouse CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein at least one of the extracellular domains comprises the “stalk region” of CD40L. The stalk region of CD40L is very long in comparison to some of the other TNFSF members (i.e., GITRL and OX40L), therefore in some embodiments the extracellular domain(s) of human CD40L comprise only a short fragment of the stalk region. In some embodiments, the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) adjacent to the TNF homology domain. In some embodiments, the fragment of the stalk region of CD40L is MQKGDQ (SEQ ID NO:98). In some embodiments, the fragment of the stalk region of CD40L is FEMQKGDQ (SEQ ID NO:99), EMQKGDQ (SEQ ID NO:100), QKGDQ (SEQ ID NO:101), or KGDQ (SEQ ID NO:102). As used herein, the stalk regions consist of amino acid sequences of CD40L, i.e., these stalk regions do not comprise any exogenous amino acids, such as an exogenous linker. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L, wherein each extracellular domain comprises a fragment of the stalk region of CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L, wherein the second and third extracellular domains include a fragment of the stalk region of CD40L. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise any peptide linkers. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the first copy and the second copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof. In some embodiments, the polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof, wherein the polypeptide does not comprise an exogenous peptide linker between the second copy and the third copy of the extracellular domain of human CD40L or a CD40-binding fragment thereof.

In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 113 to 261 of human CD40L. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 111 to 261 of human CD40L. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 112 to 261 of human CD40L. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 114 to 261 of human CD40L. In some embodiments, the agent (e.g., a polypeptide) comprises approximately amino acids 115 to 261 of human CD40L. In some embodiments, the polypeptide or agent comprises approximately amino acids 113 to 261, 111 to 261, 112 to 261, 114 to 261, or 115 to 261 of human CD40L, wherein the sequence of human CD40L is UniProt No. P29965. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:84 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:84 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:84. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:103 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:103 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:103. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:104 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:104 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:104. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:105 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:105 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:105. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:106 or a fragment thereof. In some embodiments, the polypeptide or agent comprises at least two copies of SEQ ID NO:106 or a fragment thereof. In some embodiments, the polypeptide or agent comprises three copies of SEQ ID NO:106. In some embodiments, the polypeptide or agent comprises SEQ ID NO:85. In some embodiments, the polypeptide or agent comprises SEQ ID NO:97. In some embodiments, the polypeptide or agent is a fusion polypeptide or fusion protein. In some embodiments, the fusion protein comprises a non-CD40L polypeptide (i.e., a heterologous protein). In some embodiments, the fusion polypeptide comprises a Fc region. In some embodiments, the non-CD40L polypeptide comprises a Fc region. In some embodiments, the Fc region is from an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the Fc region is selected from the group consisting of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. In some embodiments, the non-CD40L polypeptide comprises an immunoglobulin heavy chain. In some embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and light chain form an antigen-binding site. In some embodiments, the non-CD40L polypeptide comprises a single chain antibody or a Fab.

In some embodiments, the agent or polypeptide comprises SEQ ID NO:89 or SEQ ID NO:90. In some embodiments, the agent or polypeptide comprises SEQ ID NO:91 or SEQ ID NO:92.

In an additional aspect, the invention provides a polypeptide having at least about 90% sequence identity to SEQ ID NO:85 or SEQ ID NO:97, as well as polypeptides or agents comprising such a polypeptide. In some embodiments, the polypeptides or agents comprise a polypeptide consisting of SEQ ID NO:85 or SEQ ID NO:97.

In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the agent or polypeptide is monovalent. In some embodiments, the agent is bivalent. In some embodiments, the agent or polypeptide is monospecific. In some embodiments, the agent or polypeptide is bispecific.

In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the agent or polypeptide is a bispecific agent. In some embodiments, the bispecific agent is a homodimeric protein. In some embodiments, the homodimer bispecific agent comprises a polypeptide comprising a heavy chain immunoglobulin and a TNFSF trimer. In some embodiments, the heavy chain immunoglobulin is associated with a light chain to form an antigen-binding site. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain TNFSF trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain GITRL trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain OX40L trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain CD40L trimer. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds a tumor antigen. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, or TIM3. In some embodiments, the homodimeric bispecific agent binds GITR and PD-1. In some embodiments, the homodimeric bispecific agent binds GITR and PD-L1. In some embodiments, the homodimeric bispecific agent binds OX40 and PD-1. In some embodiments, the homodimeric bispecific agent binds OX40 and PD-L1. In some embodiments, the homodimeric bispecific agent binds CD40 and PD-1. In some embodiments, the homodimeric bispecific agent binds CD40 and PD-L1.

In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the agent or polypeptide is a heterodimeric bispecific agent. In some embodiments, the bispecific agent comprises a first arm which binds a member of the TNFR superfamily and a second arm which binds a second target. In some embodiments, the bispecific agent comprises a first arm which binds GITR and a second arm which binds a second target. In some embodiments, the bispecific agent comprises a first arm which binds OX40 and a second arm which binds a second target. In some embodiments, the bispecific agent comprises a first arm which binds CD40 and a second arm which binds a second target. In some embodiments, the bispecific agent comprises a first arm which binds GITR and a second arm which comprises an antigen-binding site from an antibody. In some embodiments, the bispecific agent comprises a first arm which binds OX40 and a second arm which comprises an antigen-binding site from an antibody. In some embodiments, the bispecific agent comprises a first arm which binds CD40 and a second arm which comprises an antigen-binding site from an antibody. In some embodiments, the bispecific agent comprises a second arm wherein the antigen-binding site specifically binds a tumor antigen. In some embodiments, the bispecific agent comprises a second arm wherein the antigen-binding site specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, or TIM3. In some embodiments, the bispecific agent comprises a first arm which binds GITR and a second arm which comprises an immune response stimulating agent. In some embodiments, the bispecific agent comprises a first arm which binds OX40 and a second arm which comprises an immune response stimulating agent. In some embodiments, the bispecific agent comprises a first arm which binds CD40 and a second arm which comprises an immune response stimulating agent. In some embodiments, the immune response stimulating agent may be an agonist of a target. In some embodiments, the immune response stimulating agent may be an antagonist of a target. In some embodiments, the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

In some embodiments, the bispecific agent comprises two arms, wherein each arm comprises a human CH3 domain, wherein each CH3 domain is modified to promote formation of heterodimers. In some embodiments, the first and second CH3 domains are modified using a knobs-into-holes technique. In some embodiments, the first and second CD3 domains are modified based upon electrostatic effects. In some embodiments, the bispecific agent comprises two arms, wherein the first arm comprises a first human IgG1 constant region with amino acids substitutions at positions corresponding to positions 253 and 292 of SEQ ID NO:15, wherein the amino acids are replaced with glutamate or aspartate, and the second arm comprises a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of SEQ ID NO:15, wherein the amino acids are replaced with lysine. In some embodiments, the two arms comprise a Fc region selected from the group consisting of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. In some embodiments, the bispecific agent comprises two arms, wherein the first arm comprises a first human IgG2 constant region with amino acids substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:16, wherein the amino acids are replaced with glutamate or aspartate, and the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:16, wherein the amino acids are replaced with lysine. In some embodiments, the two arms comprise a Fc region selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31.

In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the agent or polypeptide induces, activates, promotes, increases, enhances, and/or prolongs an immune response. In some embodiments, the agent increases Th1-type immune responses. In some embodiments, the agent or polypeptide increases cell-mediated immunity. In some embodiments, the agent or polypeptide increases T-cell activity. In some embodiments, the agent or polypeptide increases cytolytic T-cell (CTL) activity. In some embodiments, the agent or polypeptide increases natural killer (NK) cell activity. In some embodiments, the agent or polypeptide decreases regulatory T-cell (Treg) activity. In some embodiments, the agent or polypeptide decreases myeloid-derived suppressor cell (MDSC) activity. In some embodiments, the agent or polypeptide increases the number or the percentage of memory T-cells. In some embodiments, the agent or polypeptide increases or enhances an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, the agent or polypeptide increases or enhances an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm. In some embodiments, the agent or polypeptide is an agonist of GITRL-mediated signaling. In some embodiments, the agent or polypeptide is an agonist of GITR signaling. In some embodiments, the agent or polypeptide is an agonist of OX40L-mediated signaling. In some embodiments, the agent or polypeptide is an agonist of OX40 signaling. In some embodiments, the agent or polypeptide is an agonist of CD40L-mediated signaling. In some embodiments, the agent or polypeptide is an agonist of CD40 signaling.

In another aspect, the invention provides compositions comprising a polypeptide or agent described herein. Methods of using a composition comprising a polypeptide or agent described herein are also provided.

In another aspect, the invention provides pharmaceutical compositions comprising a polypeptide or agent described herein and a pharmaceutically acceptable carrier. Methods of treating cancer and/or inhibiting tumor growth in a subject (e.g., a human) comprising administering to the subject an effective amount of a composition comprising a polypeptide or agent described herein are also provided. Methods of treating a viral infection in a subject (e.g., a human) comprising administering to the subject an effective amount of a composition comprising a polypeptide or agent described herein are also provided.

In certain embodiments of each of the aforementioned aspects, as well as other aspects and/or embodiments described elsewhere herein, the agent or polypeptide is isolated. In certain embodiments, the agent or polypeptide is substantially pure.

In another aspect, the invention provides polynucleotides comprising a polynucleotide that encodes a polypeptide or agent described herein. In some embodiments, the polynucleotide is isolated. In some embodiments, the invention provides vectors that comprise the polynucleotides, as well as cells that comprise the vectors and/or the polynucleotides. In some embodiments, the invention also provides cells comprising or producing a polypeptide or agent described herein. In some embodiments, the cell is a monoclonal cell line.

In another aspect, the invention provides methods of modulating the immune response of a subject. In some embodiments, the invention provides a method of inducing an immune response in a subject comprising administering a polypeptide or agent described herein. In some embodiments, the invention provides a method of activating an immune response in a subject comprising administering a polypeptide or agent described herein. In some embodiments, the invention provides a method of promoting an immune response in a subject comprising administering a polypeptide or agent described herein. In some embodiments, the invention provides a method of increasing an immune response in a subject comprising administering a polypeptide or agent described herein. In some embodiments, the invention provides a method of enhancing an immune response in a subject comprising administering a polypeptide or agent described herein. In some embodiments, the invention provides a method of prolonging an immune response in a subject comprising administering a polypeptide or agent described herein. In some embodiments, the immune response is to an antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor or a tumor cell. In some embodiments, the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is a virus. In some embodiments, the antigenic stimulation is a virally-infected cell.

In some embodiments, the invention provides a method of increasing the activity of immune cells. In some embodiments, the invention provides a method of increasing the activity of immune cells comprising contacting the cells with an effective amount of a polypeptide or agent described herein. In some embodiments, the immune cells are T-cells, NK cells, monocytes, macrophages, antigen-presenting cells (APCs), and/or B-cells. In some embodiments, the invention provides a method of increasing the activity of NK cells in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of increasing the activity of T-cells in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of increasing the activity of CD4+ and/or CD8+ T-cells in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of increasing the activity of CTLs in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of increasing the activation of T-cells, CTLs, and/or NK cells in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of increasing the T-cell response in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of inhibiting the activity of Tregs in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of inhibiting the suppressive activity of Tregs in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of inhibiting the activity of MDSCs in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of inhibiting the suppressive activity of MDSCs in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of inducing an immune response in a subject without causing substantial side effects and/or immune-based toxicities comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides a method of inducing an immune response in a subject without causing cytokine release syndrome or a cytokine storm comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein.

In another aspect, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent that binds human GITR. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent that activates or enhances GITR signaling. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent that binds human OX40. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent that activates or enhances OX40 signaling. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent that binds human CD40. In some embodiments, the invention provides methods of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent that activates or enhances CD40 signaling. In some embodiments, the immune response is against a tumor cell, a tumor or cancer. In some embodiments, the immune response is against a viral infection, a viral antigen, or a virally-infected cell.

In some embodiments, the invention provides a method of increasing T-cell activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the invention provides a method of increasing CTL activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the invention provides a method of increasing NK activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the invention provides a method of decreasing or inhibiting Treg activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the invention provides a method of decreasing or inhibiting MDSC activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the TNFSF protein is GITRL. In some embodiments, the TNFSF protein is OX40L. In some embodiments, the TNFSF protein is CD40L. In some embodiments of the methods described herein, the subject has cancer.

In some embodiments, the invention provides a method of increasing T-cell activity in a subject, comprising administering to the subject a therapeutically effective amount of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, the invention provides a method of increasing CTL activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, the invention provides a method of increasing NK activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, the invention provides a method of decreasing or inhibiting Treg activity in a subject, comprising administering to the subject a therapeutically effective amount of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, the invention provides a method of decreasing or inhibiting MDSC activity in a subject, comprising administering to the subject a therapeutically effective amount of the polypeptide of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments of the methods described herein, the subject has cancer.

In another aspect, the invention provides a method of enhancing the antigen-specific memory response to a tumor. In some embodiments, a method of enhancing the antigen-specific memory response to a tumor comprises administering to a subject a therapeutically effective amount of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, a method of enhancing the antigen-specific memory response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the TNFSF protein is GITRL. In some embodiments, the TNFSF protein is OX40L. In some embodiments, the TNFSF protein is CD40L.

In another aspect, the invention provides a method of activating or enhancing a persistent or long-term immune response to a tumor. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the TNFSF protein is GITRL. In some embodiments, the TNFSF protein is OX40L. In some embodiments, the TNFSF protein is CD40L.

In another aspect, the invention provides a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of any of the polypeptides, agents, and/or bispecific agents described herein. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human TNFSF protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, the TNFSF protein is GITRL. In some embodiments, the TNFSF protein is OX40L. In some embodiments, the TNFSF protein is CD40L.

In another aspect, the invention provides methods of inhibiting tumor growth comprising contacting a tumor or tumor cell with an effective amount of a polypeptide or agent described herein. In some embodiments, a method of inhibiting growth of a tumor comprises contacting a tumor or tumor cell with an effective amount of a polypeptide or agent that binds human GITR. In some embodiments, a method of inhibiting growth of a tumor comprises contacting a tumor or tumor cell with an effective amount of a polypeptide or agent that binds human OX40. In some embodiments, a method of inhibiting growth of a tumor comprises contacting a tumor or tumor cell with an effective amount of a polypeptide or agent that binds human CD40. In some embodiments, a method of inhibiting growth of a tumor comprises contacting a tumor microenvironment with an effective amount of a polypeptide or agent that binds human GITR. In some embodiments, a method of inhibiting growth of a tumor comprises contacting a tumor microenvironment with an effective amount of a polypeptide or agent that binds human OX40. In some embodiments, a method of inhibiting growth of a tumor comprises contacting a tumor microenvironment with an effective amount of a polypeptide or agent that binds human CD40.

In another aspect, the invention provides methods of inhibiting tumor growth in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent that binds human GITR. In some embodiments, a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent that binds human OX40. In some embodiments, a method of inhibiting growth of a tumor in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent that binds human CD40.

In another aspect, the invention provides methods of treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent that binds GITR. In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent that binds OX40. In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent that binds CD40.

In another aspect, the invention provides methods of stimulating a protective response in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein in combination with an antigen of interest. In some embodiments, the antigen of interest is a tumor antigen or tumor-associated antigen (TAA). In some embodiments, the antigen of interest is a cancer cell biomarker. In some embodiments, the antigen of interest is a cancer stem cell marker.

In some embodiments of each of the aforementioned aspects and embodiments, as well as other aspects and embodiments described herein, the methods further comprise administering to the subject at least one additional therapeutic agent. In some embodiments, the at least one additional therapeutic agent is a chemotherapeutic agent. In some embodiments, the at least one additional therapeutic agent is a second immune response stimulating agent. In some embodiments, the at least one additional therapeutic agent is a checkpoint inhibitor. In some embodiments, the immune response stimulating agent is selected from the group consisting of GM-CSF, M-CSF, G-CSF, IL-3, IL-12, IL-1, IL-2, B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody. In some embodiments, the additional therapeutic agent is an anti-PD-L1 antibody.

Where aspects or embodiments of the invention are described in terms of a Markush group or other grouping of alternatives, the present invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, and also the main group absent one or more of the group members. The present invention also envisages the explicit exclusion of one or more of any of the group members in the claimed invention.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1F. (FIG. 1A) Shown is a representative drawing depicting the membrane anchored trimer topology of human GITRL. (FIG. 1B) The amino acid sequence of human GITRL is shown (SEQ ID NO:1). The cytoplasmic region, signal-anchor region, and the extracellular domain are marked. The “stalk region” within the extracellular domain is underlined. (FIG. 1C) A schematic depiction of native GITRL. (FIG. 1D) A schematic depiction of a membrane bound single chain GITRL trimer comprising the signal-anchor region of GITRL and three copies of the extracellular domain of GITRL. (FIG. 1E) A schematic depiction of a soluble single chain GITRL trimer comprising three copies of the extracellular domain of GITRL. (FIG. 1F) A schematic depiction of a soluble single chain GITRL trimer-Fc fusion polypeptide comprising three copies of the extracellular domain of GITRL linked to a Fc region.

FIG. 2. FACS analysis of a membrane bound single chain GITRL trimer and soluble GITR. HEK293 cells were transiently transfected with an expression vector encoding a membrane bound single chain human GITRL trimer and a second expression vector encoding green fluorescent protein (GFP). Transfected cells were incubated with soluble human GITR-Fc fusion protein and analyzed by flow cytometry. Positive binding is determined by observation of cells within the inset box of the FACS plot.

FIG. 3. Schematic depictions of some of the molecules proposed that contain one or more single chain GITRL trimers. These depictions are representative of additional molecules proposed that contain one or more single chain TNFSF ligand trimers.

FIG. 4. FACS analysis of a membrane-bound GITR and soluble single chain GITRL trimer-Fc fusion polypeptides. HEK293 cells were transiently transfected with an expression vector encoding full-length mouse GITR and GFP. Transfected cells were incubated with mouse GITRL fusion polypeptides 336B1, 336B2, 336B3, 336B4, 336B6, 336B10, non-GITRL fusion polypeptide 336B5, and anti-GITR antibody DTA-1 and analyzed by flow cytometry. Positive binding is determined by observation of cells within the inset box of the FACS plot.

FIG. 5. Dose response of binding of murine GITRL trimer and human GITRL trimer fusion polypeptides to GITR. Shown is a flow cytometry binding analysis of single chain murine GITRL trimer with murine IgG2a Fc domain and single chain human GITRL trimer with human IgG1 Fc domain. HEK293 cells were transiently transfected with an expression vector encoding either human or murine GITR and GFP. Transfected cells were incubated with the indicated fusion protein and analyzed by flow cytometry. Positive binding is determined by observation of cells within the inset box of the FACS plot.

FIG. 6. Activation of GITR signaling. Shown is an analysis of the ability of single chain mGITRL trimer-Fc fusion proteins to activate mGITR signaling. HEK293 cells stably transfected with a NF-κB-luciferase reporter gene and mGITR cDNA were plated into a 96-well plate and incubated with single chain mouse GITRL trimer-Fc fusion 336B3, single chain mouse GITRL trimer-Fc fusion 336B6, anti-mGITR antibody DTA-1, or control antibody. After 24 hours luciferase activity was determined. DTA-1 is an agonist antibody and was included as a positive control.

FIG. 7. Activation of GITR signaling. Shown is an analysis of the ability of single chain hGITRL trimer-Fc fusion proteins to activate hGITR signaling. HEK293 cells stably transfected with a NF-κB-luciferase reporter gene and GITR cDNA were plated into a 96-well plate and incubated with single chain human GITRL trimer-Fc fusion 336B11. After 24 hours luciferase activity was determined.

FIGS. 8A and 8B Inhibition of tumor growth by single chain GITRL trimer-Fc fusion polypeptide. The murine colon tumor line CT26.WT was implanted subcutaneously into Balb/c mice (n=10 mice/group). Mice were injected on days 7, 10, 14, and 17 with 0.25 mg/mouse of single chain mGITRL trimer-Fc fusion protein 336B3, anti-mGITR antibody DTA-1, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm³) over days post injection. (FIG. 8A) The mean values±SEM for each group. (FIG. 8B) The tumor volumes of each individual mouse from the group treated with 336B3 and the group treated with DTA-1.

FIGS. 9A and 9B. ELISpot assays for IFN-gamma and IL-10. Cells were harvested from the spleens of CT26.WT-tumor bearing mice treated with anti-mGITR antibody DTA-1, mGITRL trimer-Fc 336B3, or a control. (FIG. 9A) The number of cells producing IFN-gamma is shown. (FIG. 9B) The number of cells producing IL-10 is shown.

FIG. 10. NK cell cytotoxicity assay. Cells were harvested from the spleens of CT26.WT-tumor bearing mice treated with anti-mGITR antibody DTA-1, mGITRL trimer-Fc 336B3, or a control. CT26.WT target cells were labeled with 10 μM calcein AM mixed with the splenocytes at an E:T ratio of 25:1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.

FIG. 11. Regulatory T-cell (Treg) assay. Naïve T-cells were purified from the spleens of untreated mice. These purified T-cells were labeled with 5 μM violet tracking dye. 2×10⁵ VTD-labeled T-cells were incubated with anti-CD3 and anti-CD28 antibody-coated beads to stimulate cell proliferation. Tregs were isolated from the spleens of CT26WT tumor-bearing mice treated with mGITRL trimer-Fc 336B3, anti-mGITR antibody DTA-1, or control using a mouse Treg isolation kit. To determine the impact of Tregs on T-cell proliferation, the stimulated VTD-labeled T-cells (effectors) were co-cultured with the isolated splenic Tregs at an effector:Treg ratio of 1:0.5. On day 4, cells were washed, and incubated with anti-mouse CD4 or anti-mouse CD8 antibodies. Cells were evaluated by FACS analysis using a BD FACSCanto II instrument and BD FACSDiva software v6.1.3.

FIG. 12. Myeloid-derived suppressor cell (MDSC) assay. Naïve T-cells were purified from the spleens of untreated mice. These purified T-cells were labeled with 5 μM violet tracking dye. 2×10⁵ VTD-labeled T-cells were incubated with anti-CD3 and anti-CD28 antibody-coated beads to stimulate cell proliferation. MDSCs were isolated from the spleens of CT26.WT tumor-bearing mice treated with mGITRL trimer-Fc 336B3, anti-mGITR antibody DTA-1, or control using a mouse MDSC isolation kit. The stimulated VTD-labeled T-cells (effectors) were co-cultured with the isolated splenic MDSCs at an effector:MDSC ratio of 1:1). On day 4, cells were washed, and incubated with anti-mouse CD4 or anti-mouse CD8 antibodies. Cells were evaluated by FACS analysis using a BD FACSCanto II instrument and BD FACSDiva software v6.1.3.

FIG. 13. Inhibition of tumor growth by single chain GITRL trimer-Fc fusion polypeptide. Renca cells were implanted subcutaneously into Balb/c mice (n=10 mice/group). Mice were injected on days 7, 11, and 14 with 0.25 mg/mouse of single chain mGITRL trimer-Fc fusion protein 336B3, anti-mGITR antibody DTA-1, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data is shown as tumor volume (mm³) over days post injection.

FIGS. 14A and 14B. Cell cytotoxicity assay. (FIG. 14A) Natural killer cell activity. Cells were harvested from the spleens of Renca-tumor bearing mice treated with anti-mGITR antibody DTA-1, mGITRL trimer-Fc 336B3, or a control. YAC-1 target cells were labeled with 10 μM calcein AM mixed with the splenocytes at an E:T ratio of 25:1 and 50:1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. (FIG. 14B) T-cell cytotoxicity assay. Cells were harvested from the spleens of Renca-tumor bearing mice treated with anti-mGITR antibody DTA-1, mGITRL trimer-Fc 336B3, or a control. Renca target cells were labeled with 10 μM calcein AM mixed with the splenocytes at an E:T ratio of 25:1. Supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm.

FIGS. 15A-15G Inhibition of tumor growth by single chain GITRL trimer-Fc fusion polypeptide—a dose study. The murine colon tumor line CT26.WT was implanted subcutaneously into Balb/c mice (n=10 mice/group). Mice were treated with 30, 12.5, 6.25, 3, or 0.5 mg/kg of mGITRL trimer-Fc 336B3 or were untreated. Mice were dosed by intraperitoneal injection twice a week for a total of 6 doses. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. (FIG. 15A-15F) The tumor volumes of individual mice within each treatment group. (FIG. 15G) The average tumor volume of each treatment group.

FIGS. 16A-16G Inhibition of tumor growth by single chain GITRL trimer-Fc fusion polypeptide—a dose study. The murine colon tumor line CT26.WT was implanted subcutaneously into Balb/c mice (n=10 mice/group). Mice were treated with 2.5 mg/kg of mGITRL trimer-Fc 336B3, 12.5 mg/kg of mGITRL trimer-Fc, or were untreated (n=10 per group). Mice were treated with a single dose at 2.5 mg/kg, treated once every 2 weeks at 2.5 mg/kg, treated once every week at 2.5 mg/kg, treated twice a week at 2.5 mg/kg, or treated twice a week at 12.5 mg/kg for only 3 doses. (FIG. 16A-16F) The tumor volumes of individual mice within each treatment group. (FIG. 16G) The average tumor volume of each treatment group.

FIGS. 17A-17F. In vivo tumor growth inhibition by single chain GITRL trimer-Fc protein in immune cell-depleted mice. For in vivo depletion of specific cell populations, Balb/c mice were given an intraperitoneal injection of anti-CD4 antibody (500 ug/dose), anti-CD8 antibody (500 ug/dose), a combination of anti-CD4 and anti-CD8 antibodies (500 ug/dose each), anti-asialo GM-1 antibody (25 ul), or a control IgG2 antibody (LFT-2; 500 ug/dose) 2 days and 1 day prior to tumor cell implantation, and then additional injections were given 1 day after implantation and twice a week during the experiment. The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in the cell-depleted mice. Mice were treated with 0.25 mg/mouse of mGITRL trimer-Fc 336B3 or a control antibody (n=10 per group). Mice were dosed by intraperitoneal injection twice a week. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. (FIG. 17A-17E) The tumor volumes of individual mice within each treatment group. (FIG. 17F) The average tumor volume of each treatment group.

FIGS. 18A-18D. In vivo tumor growth inhibition by single chain GITRL trimer-Fc protein and anti-PD-1 antibody. The murine adenocarcinoma cell line Renca was implanted subcutaneously (5×10⁵ cells/mouse) in Balb/c mice. Mice were treated with 12.5 mg/kg of single chain mGITRL trimer-Fc 336B3, an anti-PD-1 antibody, a combination of 336B3 and anti-PD-1 antibody, or a control antibody (n=20 per group). Mice were administered 336B3 by intraperitoneal injection twice a week for only 3 doses and anti-PD-1 antibody was administered twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. (FIG. 18A-18D) The tumor volumes of individual mice within each treatment group.

FIGS. 19A-19E. In vivo tumor growth inhibition by single chain GITRL trimer-Fc protein and anti-PD-L1 antibody. The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice. Mice were treated with 0.25 mg/mouse of single chain mGITRL trimer-Fc 336B3, an anti-PD-L1 antibody, a combination of 336B3 and anti-PD-L1 antibody, or a control antibody (n=10-20 per group). Mice were administered 336B3 by intraperitoneal injection twice a week for only 3 doses and anti-PD-L1 antibody was administered twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. FIG. 19A-19D. The tumor volumes of individual mice within each treatment group. FIG. 19E. Tumor growth after re-challenge with CT26.WT cells.

FIGS. 20A-20E. In vivo tumor growth inhibition by single chain GITRL trimer-Fc protein and anti-PD-1 antibody. The murine melanoma cell line B16-F10 cells were implanted subcutaneously (5000 cells/mouse) in C57BL/6 mice. Mice were treated with 5 mg/kg of single chain mGITRL trimer-Fc 336B3, 10 mg/kg of an anti-mPD-1 antibody, a combination of 336B3 and anti-mPD-1 antibody, or a control antibody (n=10 per group). Mice were administered 336B3, anti-mPD-1 antibody, or control antibody by intraperitoneal injection twice a week. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. (FIG. 20A-20D) The tumor volumes of individual mice within each treatment group. (FIG. 20E) The average tumor volume of each treatment group.

FIGS. 21A-21E Inhibition of tumor growth by single chain GITRL trimer-Fc and OX40L trimer-Fc fusion polypeptide. The murine colon tumor line CT26.WT was implanted subcutaneously into Balb/c mice (n=10 mice/group). Mice were injected twice a week for 3 doses with 0.25 mg/mouse of single chain mOX40L trimer-Fc fusion protein, anti-mOX40 antibody, mGITRL trimer-Fc, anti-mGITR antibody DTA-1, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. FIG. 21A. The tumor volumes of each individual mouse from the group treated with control antibody. FIG. 21B. The tumor volumes of each individual mouse from the group treated with mOX40L-Fc. FIG. 21C. The tumor volumes of each individual mouse from the group treated with agonist anti-OX40 antibody. FIG. 21D. Average tumor volume of three groups of mice. FIG. 21E. Average tumor volume of five groups of mice. Data is shown as tumor volume (mm³) over days post injection.

FIG. 22. Inhibition of tumor growth by single chain GITRL trimer-Fc fusion polypeptide—a dose study in B16-B10 mouse model. B16-F10 cells were injected subcutaneously in C57BL/6 mice and were allowed to reach an average size of approximately 84 mm³. Mice were treated with 0.5 mg/kg of anti-mGITR antibody DTA-1, 30, 10, 2.5, 0.5, and 0.05 mg/kg of single chain mGITRL trimer-Fc 336B3, two different control antibodies, or saline (n=10 per group). Mice were administered 336B3, antibodies, or saline by intraperitoneal injection once a week. Tumor growth was monitored and tumor volumes were measured with electronic calipers. The results as shown percent of tumors that were less than 300 mm³ in each treatment group.

FIG. 23A-23L. Cytokine production. The murine colon tumor line CT26.WT was implanted subcutaneously into Balb/c mice (n=10 mice/group). Mice were injected twice a week for 3 doses with 0.25 mg/mouse of single chain mOX40L trimer-Fc fusion protein, anti-mOX40 antibody, mGITRL trimer-Fc, anti-mGITR antibody DTA-1, or a control antibody. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Cells were harvested on Day 26 from the spleens of the mice in each treatment group. The cells were cultured in the presence or the absence of the tumor specific CD8⁺ T-cell peptide AH-1. After 48 hours, cytokine levels in cell supernatants were measured using a multiplex panel for the Luminex® platform (ThermoFisher Scientific) following the manufacturer's instructions. FIG. 23A. IL-2. FIG. 23B. IL-4. FIG. 23C. IL-5. FIG. 23D. IL-6. FIG. 23E. IL-10. FIG. 23F. IL-13. FIG. 23G. MIP-1b. FIG. 23H. FasL—The cytokine levels for the control, OX40L-Fc and gITRL-Fc were below the limits of the assay. FIG. 23I. GM-CSF. FIG. 23J. sCD137. FIG. 23K. IFN-gamma. FIG. 23L. Granzyme B.

FIG. 24A-24D. Inhibition of tumor growth by single chain GITRL trimer-Fc protein. CT26.WT cells were implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and allowed to grow until tumors were an average size of approximately 300 mm³. Mice were treated with 0.25 mg/mouse of single chain mGITRL trimer-Fc 336B3, agonist anti-GITR antibody DTA-1, or a control antibody (n=17 per group). Mice were dosed twice a week for a total of 3 doses by intraperitoneal injection. Tumor growth was monitored and tumor volumes were measured with electronic calipers. As possible, mice were followed beyond 80 days to assess long-term survival. FIG. 24A. The tumor volumes of each individual mouse from the group treated with control antibody. FIG. 24B. The tumor volumes of each individual mouse from the group treated with mGITRL-Fc. FIG. 24C. The tumor volumes of each individual mouse from the group treated with agonist anti-GITR antibody DTA-1. FIG. 24D. Survival curve of all treated groups.

FIG. 25. Inhibition of tumor growth by single chain GITRL trimer-Fc protein in a humanized mouse model. Humanized mice were injected subcutaneously with patient-derived melanoma tumor cells (OMP-M9, 75,000 cells/mouse). Tumors were allowed to grow 16 days until they had reached an average volume of approximately 60 mm³. Tumor-bearing mice were randomized into 2 groups (n=3 mice per group). Tumor-bearing mice were treated with either a control protein or hGITRL trimer-Fc OMP-336B11. Mice were dosed twice weekly at 10 mg/kg. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel agents, including, but not limited to, polypeptides, soluble proteins, fusion proteins, homodimeric bispecific molecules, and heterodimeric bispecific molecules that modulate the immune response. The agents include agonists and antagonists of receptors and ligands that are members of the TNF superfamily involved in cell interactions and immune response signaling. Related polypeptides and polynucleotides, compositions comprising the agents, and methods of making the agents are also provided. Methods of screening for agents that modulate the immune response are provided. Methods of using the novel agents, such as methods of activating an immune response, methods of stimulating an immune response, methods of promoting an immune response, methods of increasing an immune response, methods of activating NK cells, methods of activating T-cells, including CTLs, methods of increasing the activity of NK cells, methods of increasing the activity of T-cells, including CTLs, methods of promoting the activity of NK cells, methods of promoting the activity of T-cells, including CTLs, methods of inhibiting the activity of Tregs, methods of inhibiting the activity of MDSCs, methods of inhibiting tumor growth, methods of treating cancer, and/or methods of treating viral diseases are provided. Methods of inhibiting an immune response, methods of suppressing an immune response, methods of decreasing activity of T-cells, and/or methods of treating autoimmune diseases are further provided.

I. DEFINITIONS

To facilitate an understanding of the present invention, a number of terms and phrases are defined below.

The terms “agonist” and “agonistic” as used herein refer to or describe a polypeptide or agent that is capable of, directly or indirectly, substantially inducing, activating, promoting, increasing, or enhancing the biological activity of a target and/or a pathway. The term “agonist” is used herein to include any agent that partially or fully induces, activates, promotes, increases, or enhances the activity of a protein or other target of interest.

The terms “antagonist” and “antagonistic” as used herein refer to or describe a polypeptide or agent that is capable of, directly or indirectly, partially or fully blocking, inhibiting, reducing, or neutralizing a biological activity of a target and/or pathway. The term “antagonist” is used herein to include any agent that partially or fully blocks, inhibits, reduces, or neutralizes the activity of a protein or other target of interest.

The terms “modulation” and “modulate” as used herein refer to a change or an alteration in a biological activity. Modulation includes, but is not limited to, stimulating an activity or inhibiting an activity. Modulation may be an increase in activity or a decrease in activity, a change in binding characteristics, or any other change in the biological, functional, or immunological properties associated with the activity of a protein, a pathway, a system, or other biological targets of interest.

The term “soluble protein” as used herein refers to a protein or a fragment thereof that can be secreted from a cell in soluble form.

The term “fusion protein” or “fusion polypeptide” as used herein refers to a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.

The term “linker” or “linker region” as used herein refers to a linker inserted between a first polypeptide (e.g., copies of a GITRL extracellular domain or fragments thereof) and a second polypeptide (e.g., a Fc region). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the polypeptides. Preferably, linkers are not antigenic and do not elicit an immune response.

The term “antibody” as used herein refers to an immunoglobulin molecule that recognizes and specifically binds a target, such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of any of the foregoing, through at least one antigen-binding site wherein the antigen-binding site is usually within the variable region of the immunoglobulin molecule. As used herein, the term encompasses intact polyclonal antibodies, intact monoclonal antibodies, antibody fragments (such as Fab, Fab′, F(ab′)2, and Fv fragments), single chain Fv (scFv) antibodies, multispecific antibodies, bispecific antibodies, monospecific antibodies, monovalent antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising an antigen-binding site of an antibody, and any other modified immunoglobulin molecule comprising an antigen-binding site as long as the antibodies exhibit the desired biological activity. An antibody can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), based on the identity of their heavy-chain constant domains referred to as alpha, delta, epsilon, gamma, and mu, respectively. The different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules, including but not limited to, toxins and radioisotopes.

The term “antibody fragment” refers to a portion of an intact antibody and refers to the antigenic determining variable regions of an intact antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab′, F(ab′)2, and Fv fragments, linear antibodies, single chain antibodies, and multispecific antibodies formed from antibody fragments. “Antibody fragment” as used herein comprises an antigen-binding site or epitope-binding site.

The term “variable region” of an antibody refers to the variable region of an antibody light chain, or the variable region of an antibody heavy chain, either alone or in combination. Generally, the variable region of heavy and light chains each consist of four framework regions (FR) and three complementarity determining regions (CDRs), also known as “hypervariable regions”. The CDRs in each chain are held together in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding sites of the antibody. There are at least two techniques for determining CDRs: (1) an approach based on cross-species sequence variability (i.e., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition, National Institutes of Health, Bethesda Md.), and (2) an approach based on crystallographic studies of antigen-antibody complexes (Al Lazikani et al., 1997, J. Mol. Biol., 273:927-948). In addition, combinations of these two approaches are sometimes used in the art to determine CDRs.

The term “monoclonal antibody” as used herein refers to a homogenous antibody population involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include a mixture of different antibodies directed against different antigenic determinants. The term “monoclonal antibody” encompasses both intact and full-length monoclonal antibodies as well as antibody fragments (e.g., Fab, Fab′, F(ab′)2, Fv), single chain (scFv) antibodies, fusion proteins comprising an antibody fragment, and any other modified immunoglobulin molecule comprising an antigen-binding site. Furthermore, “monoclonal antibody” refers to such antibodies made by any number of techniques, including but not limited to, hybridoma production, phage selection, recombinant expression, and transgenic animals.

The term “humanized antibody” as used herein refers to forms of non-human (e.g., murine) antibodies that are specific immunoglobulin chains, chimeric immunoglobulins, or fragments thereof that contain minimal non-human sequences. Typically, humanized antibodies are human immunoglobulins in which residues of the CDRs are replaced by residues from the CDRs of a non-human species (e.g., mouse, rat, rabbit, or hamster) that have the desired specificity, affinity, and/or binding capability. In some instances, the Fv framework region residues of a human immunoglobulin are replaced with the corresponding residues in an antibody from a non-human species. The humanized antibody can be further modified by the substitution of additional residues either in the Fv framework region and/or within the replaced non-human residues to refine and optimize antibody specificity, affinity, and/or binding capability. The humanized antibody may comprise variable domains containing all or substantially all of the CDRs that correspond to the non-human immunoglobulin whereas all or substantially all of the framework regions are those of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin sequence. In some embodiments, the variable domains comprise the framework regions of a human immunoglobulin consensus sequence. The humanized antibody can also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. A humanized antibody is usually considered distinct from a chimeric antibody.

The term “human antibody” as used herein refers to an antibody produced by a human or an antibody having an amino acid sequence corresponding to an antibody produced by a human made using any of the techniques known in the art.

The term “chimeric antibody” as used herein refers to an antibody wherein the amino acid sequence of the immunoglobulin molecule is derived from two or more species. Typically, the variable region of both light and heavy chains corresponds to the variable region of antibodies derived from one species of mammals (e.g., mouse, rat, rabbit, etc.) with the desired specificity, affinity, and/or binding capability, while the constant regions are homologous to the sequences in antibodies derived from another species (usually human) to avoid eliciting an immune response in that species.

The terms “epitope” and “antigenic determinant” are used interchangeably herein and refer to that portion of an antigen capable of being recognized and specifically bound by a particular antibody. When the antigen is a polypeptide, epitopes can be formed both from contiguous amino acids and noncontiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed from contiguous amino acids (also referred to as linear epitopes) are typically retained upon protein denaturing, whereas epitopes formed by tertiary folding (also referred to as conformational epitopes) are typically lost upon protein denaturing. An epitope typically includes at least 3, and more usually, at least 5, 6, 7, or 8-10 amino acids in a unique spatial conformation.

The terms “selectively binds” or “specifically binds” mean that a polypeptide or agent interacts more frequently, more rapidly, with greater duration, with greater affinity, or with some combination of the above to the epitope, protein, or target molecule than with alternative substances, including related and unrelated proteins. In certain embodiments “specifically binds” means, for instance, that a polypeptide or agent binds a protein or target with a K_(D) of about 0.1 mM or less, but more usually less than about 1 μM. In certain embodiments, “specifically binds” means that a polypeptide or agent binds a target with a K_(D) of at least about 0.1 μM or less, at least about 0.01 μM or less, or at least about 1 nM or less. Because of the sequence identity between homologous proteins in different species, specific binding can include a polypeptide or agent that recognizes a protein or target in more than one species. Likewise, because of homology within certain regions of polypeptide sequences of different proteins, specific binding can include a polypeptide or agent that recognizes more than one protein or target. It is understood that, in certain embodiments, a polypeptide or agent that specifically binds a first target may or may not specifically bind a second target. As such, “specific binding” does not necessarily require (although it can include) exclusive binding, i.e. binding to a single target. Thus, a polypeptide or agent may, in certain embodiments, specifically bind more than one target. In certain embodiments, multiple targets may be bound by the same antigen-binding site on the polypeptide or agent. For example, an antibody may, in certain instances, comprise two identical antigen-binding sites, each of which specifically binds the same epitope on two or more proteins. In certain alternative embodiments, an antibody may be bispecific and comprise at least two antigen-binding sites with differing specificities. Generally, but not necessarily, reference to “binding” means “specific binding”.

The terms “polypeptide” and “peptide” and “protein” are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are, for example, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids), as well as other modifications known in the art. It is understood that, because the polypeptides of this invention may be based upon antibodies or other members of the immunoglobulin superfamily, in certain embodiments, the polypeptides can occur as single chains or as associated chains.

The terms “polynucleotide” and “nucleic acid” and “nucleic acid molecule” are used interchangeably herein and refer to polymers of nucleotides of any length, and include DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into a polymer by DNA or RNA polymerase.

The terms “identical” or percent “identity” in the context of two or more nucleic acids or polypeptides, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity may be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software that may be used to obtain alignments of amino acid or nucleotide sequences are well-known in the art. These include, but are not limited to, BLAST, ALIGN, Megalign, BestFit, GCG Wisconsin Package, and variants thereof. In some embodiments, two nucleic acids or polypeptides of the invention are substantially identical, meaning they have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and in some embodiments at least 95%, 96%, 97%, 98%, 99% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using a sequence comparison algorithm or by visual inspection. In some embodiments, identity exists over a region of the amino acid sequences that is at least about 10 residues, at least about 20 residues, at least about 40-60 residues, at least about 60-80 residues in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 residues, such as at least about 80-100 residues, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as the coding region of a target protein or an antibody. In some embodiments, identity exists over a region of the nucleotide sequences that is at least about 10 bases, at least about 20 bases, at least about 40-60 bases, at least about 60-80 bases in length or any integral value there between. In some embodiments, identity exists over a longer region than 60-80 bases, such as at least about 80-1000 bases or more, and in some embodiments the sequences are substantially identical over the full length of the sequences being compared, such as a nucleotide sequence encoding a protein of interest.

A “conservative amino acid substitution” is one in which one amino acid residue is replaced with another amino acid residue having a similar side chain Families of amino acid residues having similar side chains have been generally defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For example, substitution of a phenylalanine for a tyrosine is a conservative substitution. Generally, conservative substitutions in the sequences of the polypeptides, soluble proteins, and/or antibodies of the invention do not abrogate the binding of the polypeptide, soluble protein, or antibody containing the amino acid sequence, to the target binding site. Methods of identifying amino acid conservative substitutions which do not eliminate binding are well-known in the art.

The term “vector” as used herein means a construct, which is capable of delivering, and usually expressing, one or more gene(s) or sequence(s) of interest in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmid, cosmid, or phage vectors, DNA or RNA expression vectors associated with cationic condensing agents, and DNA or RNA expression vectors encapsulated in liposomes.

A polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is “isolated” is a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is in a form not found in nature. Isolated polypeptides, soluble proteins, antibodies, polynucleotides, vectors, cells, or compositions include those which have been purified to a degree that they are no longer in a form in which they are found in nature. In some embodiments, a polypeptide, soluble protein, antibody, polynucleotide, vector, cell, or composition which is isolated is substantially pure.

The term “substantially pure” as used herein refers to material which is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

The term “immune response” as used herein includes responses from both the innate immune system and the adaptive immune system. It includes both cell-mediated and/or humoral immune responses. It includes both T-cell and B-cell responses, as well as responses from other cells of the immune system such as natural killer (NK) cells, monocytes, macrophages, etc.

The terms “cancer” and “cancerous” as used herein refer to or describe the physiological condition in mammals in which a population of cells are characterized by unregulated cell growth. Examples of cancer include, but are not limited to, carcinoma, blastoma, sarcoma, and hematologic cancers such as lymphoma and leukemia.

The terms “tumor” and “neoplasm” as used herein refer to any mass of tissue that results from excessive cell growth or proliferation, either benign (noncancerous) or malignant (cancerous) including pre-cancerous lesions.

The term “metastasis” as used herein refers to the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.

The terms “cancer stem cell” and “CSC” and “tumor stem cell” and “tumor initiating cell” are used interchangeably herein and refer to cells from a cancer or tumor that: (1) have extensive proliferative capacity; 2) are capable of asymmetric cell division to generate one or more types of differentiated cell progeny wherein the differentiated cells have reduced proliferative or developmental potential; and (3) are capable of symmetric cell divisions for self-renewal or self-maintenance. These properties confer on the cancer stem cells the ability to form or establish a tumor or cancer upon serial transplantation into an appropriate host (e.g., a mouse) compared to the majority of tumor cells that fail to form tumors. Cancer stem cells undergo self-renewal versus differentiation in a chaotic manner to form tumors with abnormal cell types that can change over time as mutations occur.

The terms “cancer cell” and “tumor cell” refer to the total population of cells derived from a cancer or tumor or pre-cancerous lesion, including both non-tumorigenic cells, which comprise the bulk of the cancer cell population, and tumorigenic stem cells (cancer stem cells). As used herein, the terms “cancer cell” or “tumor cell” will be modified by the term “non-tumorigenic” when referring solely to those cells lacking the capacity to renew and differentiate to distinguish those tumor cells from cancer stem cells.

The term “tumorigenic” as used herein refers to the functional features of a cancer stem cell including the properties of self-renewal (giving rise to additional tumorigenic cancer stem cells) and proliferation to generate all other tumor cells (giving rise to differentiated and thus non-tumorigenic tumor cells).

The term “tumorigenicity” as used herein refers to the ability of a random sample of cells from the tumor to form palpable tumors upon serial transplantation into appropriate hosts (e.g., mice).

The term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.

The term “pharmaceutically acceptable” refers to a substance approved or approvable by a regulatory agency of the Federal government or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

The terms “pharmaceutically acceptable excipient, carrier or adjuvant” or “acceptable pharmaceutical carrier” refer to an excipient, carrier or adjuvant that can be administered to a subject, together with at least one agent of the present disclosure, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic effect. In general, those of skill in the art and the U.S. FDA consider a pharmaceutically acceptable excipient, carrier, or adjuvant to be an inactive ingredient of any formulation.

The terms “effective amount” or “therapeutically effective amount” or “therapeutic effect” refer to an amount of a polypeptide or agent described herein (e.g., a fusion protein, a soluble receptor, an antibody, a polypeptide, a polynucleotide, a small organic molecule, or other drug) effective to “treat” a disease or disorder in a subject such as, a mammal. In the case of cancer or a tumor, the therapeutically effective amount of a polypeptide or agent (e.g., polypeptide, soluble protein, or antibody) has a therapeutic effect and as such can boost the immune response, boost the anti-tumor response, increase cytolytic activity of immune cells, increase killing of tumor cells by immune cells, reduce the number of tumor cells; decrease tumorigenicity, tumorigenic frequency or tumorigenic capacity; reduce the number or frequency of cancer stem cells; reduce the tumor size; reduce the cancer cell population; inhibit or stop cancer cell infiltration into peripheral organs including, for example, the spread of cancer into soft tissue and bone; inhibit and stop tumor or cancer cell metastasis; inhibit and stop tumor or cancer cell growth; relieve to some extent one or more of the symptoms associated with the cancer; reduce morbidity and mortality; improve quality of life; or a combination of such effects.

The terms “treating” or “treatment” or “to treat” or “alleviating” or “to alleviate” refer to both (1) therapeutic measures that cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic condition or disorder and (2) prophylactic or preventative measures that prevent or slow the development of a targeted pathologic condition or disorder. Thus those in need of treatment include those already with the disorder; those prone to have the disorder; and those in whom the disorder is to be prevented. In the case of cancer or a tumor, a subject is successfully “treated” according to the methods of the present invention if the patient shows one or more of the following: an increased immune response, an increased anti-tumor response, increased cytolytic activity of immune cells, increased killing of tumor cells by immune cells, a reduction in the number of or complete absence of cancer cells; a reduction in the tumor size; inhibition of or an absence of cancer cell infiltration into peripheral organs including the spread of cancer cells into soft tissue and bone; inhibition of or an absence of tumor or cancer cell metastasis; inhibition or an absence of cancer growth; relief of one or more symptoms associated with the specific cancer; reduced morbidity and mortality; improvement in quality of life; reduction in tumorigenicity; reduction in the number or frequency of cancer stem cells; or some combination of effects.

As used in the present disclosure and claims, the singular forms “a”, “an” and “the” include plural forms unless the context clearly dictates otherwise.

It is understood that wherever embodiments are described herein with the language “comprising” otherwise analogous embodiments described in terms of “consisting of” and/or “consisting essentially of” are also provided. It is also understood that wherever embodiments are described herein with the language “consisting essentially of” otherwise analogous embodiments described in terms of “consisting of” are also provided.

As used herein, reference to “about” or “approximately” a value or parameter includes (and describes) embodiments that are directed to that value or parameter. For example, description referring to “about X” includes description of “X”.

The term “and/or” as used in a phrase such as “A and/or B” herein is intended to include both A and B; A or B; A (alone); and B (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

II. TNF RECEPTOR SUPERFAMILY BINDING AGENTS

The present invention provides agents that bind members of the TNF receptor superfamily (TNFRSF). TNFRSF members include, but may not be limited to, 4-1BB, BAFF, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL R1, DcTRAIL R2, DR3, DR6, EDA2R, EDAR, Fas (CD95), GITR, HVEM, lymphotoxin beta R, NGFR, osteoprotegerin, OX40, RANK, RELT, TACI, TNFRH3, TNF R1, TNF R2, TRAIL R1, TRAIL R2, TRAIL R3, TRAIL R4, TROY, and TWEAK R. In some embodiments, the polypeptide or agent binds glucocorticoid-induced tumor necrosis factor receptor-related protein (GITR). These agents may be referred to herein as “GITR-binding agents”. In certain embodiments, the polypeptide or agent is a GITR agonist. In certain embodiments, the polypeptide or agent induces, activates, enhances, increases, and/or prolongs GITR signaling. In some embodiments, the polypeptide or agent binds OX40. These polypeptides or agents may be referred to herein as “OX40-binding agents”. In certain embodiments, the polypeptide or agent is an OX40 agonist. In certain embodiments, the polypeptide or agent induces, activates, enhances, increases, and/or prolongs OX40 signaling. In some embodiments, the polypeptide or agent binds CD40. These polypeptides or agents may be referred to herein as “CD40-binding agents”. In certain embodiments, the polypeptide or agent is a CD40 agonist. In certain embodiments, the polypeptide or agent induces, activates, enhances, increases, and/or prolongs CD40 signaling.

In certain embodiments, the agent is a polypeptide. In certain embodiments, the agent is a soluble protein. In some embodiments, the agent is a fusion polypeptide. In some embodiments, the agent is a soluble ligand or soluble “co-receptor”. In some embodiments, the polypeptide or agent comprises a fragment of human GITRL. In some embodiments, the polypeptide or agent comprises a fragment of human OX40L. In some embodiments, the polypeptide or agent comprises a fragment of human CD40L. In some embodiments, a fragment of the extracellular domain of human GITRL, OX40L, or CD40L can demonstrate altered biological activity (e.g., increased protein half-life) compared to a soluble agent comprising the entire extracellular domain.

In some embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein. In some embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises the stalk region of the TNFSF protein. In some embodiments, the TNFSF protein is selected from the group consisting of: GITRL, OX40L, 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30 ligand, CD40 ligand (CD40L), EDA, EDA-Al, EDA-A2, Fas ligand (CD95L), LIGHT, lymphotoxin, lymphotoxin-beta, lymphotoxin-alpha, TL1A, TNF-alpha, TRAIL, TRANCE, and TWEAK.

The full-length amino acid (aa) sequence of human GITRL is known in the art (UniProt No. Q9UNG2) and is provided herein as SEQ ID NO:1. In some embodiments, a polypeptide or agent comprises at least one copy of the extracellular domain of GITRL or a GITR-binding fragment thereof. In certain embodiments, the “extracellular domain” of GITRL is approximately amino acids 71-199 of SEQ ID NO:1. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the extracellular domain of GITRL. Thus, the N-terminus and/or C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. As used herein, the extracellular domain of GITRL generally comprises the “stalk region” and the “TNF family domain”. Thus, in some embodiments, the copy of the extracellular domain of GITRL in the polypeptides or agents described herein comprises the “stalk region” of GITRL. The “stalk region” of GITRL is approximately amino acids 71-77 of SEQ ID NO:1. The stalk region comprises approximately amino acids LQLETAK (SEQ ID NO:32). The “TNF homology domain” or “TNF family domain” of GITRL is approximately amino acids 89-192 of SEQ ID NO:1. The TNF homology domain of GITRL comprises SEQ ID NO:33. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of GITRL or a GITR-binding fragment thereof. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:3. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:3. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:3. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:64. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:64. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:64. In certain embodiments, a polypeptide or agent comprises at least a first, second, and third copy of the extracellular domain of GITRL or a GITR-binding fragment thereof as a single chain polypeptide. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:5. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:5. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:5. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:5. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:5. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:5. In some embodiments, a polypeptide or agent comprises SEQ ID NO:66. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:66. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:66. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:66. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:66. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:66. In certain embodiments, a polypeptide or agent comprises at least a first, second, and third copy of a fragment of the extracellular domain of GITRL. In some embodiments, the copies of the extracellular domain of GITRL consist of the same amino acid sequence. In some embodiments, the copies of the extracellular domain of GITRL are not identical. In some embodiments, the copies of the extracellular domain of GITRL comprise substitutions, deletions, and/or additions to the amino acid sequence of human GITRL as compared to the wild-type sequence.

In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein at least one of the extracellular domains comprises the stalk region of GITRL. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein at least two of the extracellular domains comprise the stalk region of GITRL. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof, wherein each extracellular domain comprises the stalk region of GITRL. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL, wherein the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL, wherein the polypeptide does not comprise an exogenous peptide linker between any of the copies of the extracellular domain or a fragment thereof. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL, wherein each extracellular domain comprises the stalk region and the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of human GITRL or a GITR-binding fragment thereof, wherein the second and third copies of the extracellular domain comprise a stalk. In some embodiments, a polypeptide or extracellular domain of GITRL comprises amino acids 71-199 of SEQ ID NO:1. In some embodiments, the extracellular domain of GITRL comprises SEQ ID NO:3. In some embodiments, the extracellular domain of GITRL comprises SEQ ID NO:64. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:5. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:5. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:66. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:66.

The full-length amino acid (aa) sequence of human OX40L is known in the art (UniProt No. P23510) and is provided herein as SEQ ID NO:40. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain of OX40L or a fragment thereof. In certain embodiments, the “extracellular domain” of OX40L is approximately amino acids 51-183 of SEQ ID NO:40. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the extracellular domain of OX40L. Thus, the N-terminus and/or C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. As used herein, the extracellular domain of OX40L generally comprises the “stalk region” and the “TNF family domain”. Thus, in some embodiments, a copy of the extracellular domain of OX40L in the polypeptides or agents described herein comprises the “stalk region” of OX40L. The “stalk region” of OX40L is approximately amino acids 51-57 of SEQ ID NO:40. The stalk region comprises approximately amino acids QVSHRYP (SEQ ID NO:55). In some embodiments, the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain. In some embodiments, the stalk region comprises a fragment of the stalk region. In some embodiments, the stalk region comprises additional amino acids. In some embodiments, the stalk region comprises amino acids ALQVSHRYP (variant 1; SEQ ID NO:74). In some embodiments, the stalk region comprises amino acids SHRYP (variant 2; SEQ ID NO:75). In some embodiments, the stalk region comprises amino acids HRYP (variant 3; SEQ ID NO:76). The “TNF homology domain” or “TNF family domain” of OX40L is approximately amino acids 84-178 of SEQ ID NO:40. The TNF homology domain comprises SEQ ID NO:56. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:42. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:42. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:42. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:67. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:67. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:67. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:77. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:77. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:77. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:78. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:78. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:78. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:79. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:79. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:79. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of OX40L or an OX40-binding fragment thereof. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of OX40L or a fragment thereof as a single chain polypeptide. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:44. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:44. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:44. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:44. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:44. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:44. In certain embodiments, the polypeptide or agent comprises SEQ ID NO:69. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:69. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:69. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:69. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:69. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:69. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:71. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:71. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:71. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:71. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:71. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:71. In certain embodiments, a polypeptide or agent comprises at least a first, second, and third copy of a fragment of the extracellular domain of OX40L. In some embodiments, the copies of the extracellular domain of OX40L consist of the same amino acid sequence. In some embodiments, the copies of the extracellular domain of OX40L are not identical. In some embodiments, the copies of the extracellular domain of OX40L comprise substitutions, deletions, and/or additions to the amino acid sequence of human OX40L as compared to the wild-type sequence.

In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L or a fragment thereof. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L or a fragment thereof, wherein at least one of the extracellular domains comprises the stalk region. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L or a fragment thereof, wherein each extracellular domain comprises the stalk region. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L, wherein the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human OX40L, wherein each extracellular domain comprises the stalk region and the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, the extracellular domain of OX40L comprises amino acids 51-183 of SEQ ID NO:40. In some embodiments, the extracellular domain of OX40L comprises SEQ ID NO:42. In some embodiments, the extracellular domain of OX40L consists of SEQ ID NO:42. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:44. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:44. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:69. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:69.

The full-length amino acid (aa) sequence of human CD40L is known in the art (UniProt No. P29965) and is provided herein as SEQ ID NO:82. In some embodiments, the polypeptide or agent comprises at least one copy of the extracellular domain of CD40L or a fragment thereof. In certain embodiments, the “extracellular domain” of CD40L is approximately amino acids 47-261 of SEQ ID NO:82. Those of skill in the art may differ in their understanding of the exact amino acids corresponding to the extracellular domain of CD40L. Thus, the N-terminus and/or C-terminus of the extracellular domain described herein may extend or be shortened by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. As used herein, the extracellular domain of CD40L generally comprises the “stalk region” or a fragment of the stalk region and the “TNF family domain”. The stalk region of CD40L is approximately 72 amino acids, much longer than the stalk regions of GITRL and CD40L. In some embodiments, to allow for proper folding and conformation of a single chain CD40L trimer, the stalk region comprises a fragment of the CD40L stalk region more equivalent in length to the stalk regions of GITRL or OX40L. Thus, in some embodiments, a copy of the extracellular domain of CD40L in the polypeptides or agents described herein comprises a fragment of the stalk region of CD40L. In some embodiments, the stalk region comprises about 4-20 amino acids. In some embodiments, the stalk region comprises about 4-10 amino acids. In some embodiments, the stalk region comprises the amino acids (e.g., 4-10 amino acids) upstream from the TNF homology domain. The stalk region of CD40L is approximately amino acids 47-112 of SEQ ID NO:82. In some embodiments, the stalk region comprises a fragment of the CD40L stalk region. In some embodiments, fragments of the CD40L stalk region comprise MQKGDQ (SEQ ID NO:98; fragment 1); FEMQKGDQ (SEQ ID NO:99; fragment 2); EMQKGDQ (SEQ ID NO:100; fragment 3); QKGDQ (SEQ ID NO:101; fragment 4); or KGDQ (SEQ ID NO:102; fragment 5). The “TNF homology domain” or “TNF family domain” of CD40L is approximately amino acids 122-261 of SEQ ID NO:82. The TNF homology domain comprises SEQ ID NO:94. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:84. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:84. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:84. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:95. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:95. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:95. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:103. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:103. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:103. In some embodiments, a polypeptide or agent comprises at least one copy of SEQ ID NO:104. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:104. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:104. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:105. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:105. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:105. In some embodiments, the polypeptide or agent comprises at least one copy of SEQ ID NO:106. In some embodiments, a polypeptide or agent comprises at least two copies of SEQ ID NO:106. In some embodiments, a polypeptide or agent comprises three copies of SEQ ID NO:106. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of CD40L or an CD40-binding fragment thereof. In certain embodiments, a polypeptide or agent comprises a first, second, and third copy of the extracellular domain of CD40L or a fragment thereof as a single chain polypeptide. In certain embodiments, a polypeptide or agent comprises SEQ ID NO:85. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:85. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:85. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:85. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:85. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:85. In certain embodiments, the polypeptide or agent comprises SEQ ID NO:97. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 90% sequence identity to SEQ ID NO:97. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least about 95% sequence identity to SEQ ID NO:97. In certain embodiments, a polypeptide or agent comprises a polypeptide having at least 96%, 97%, 98%, 99% sequence identity to SEQ ID NO:97. In some embodiments, a polypeptide or agent comprises a polypeptide consisting essentially of SEQ ID NO:97. In some embodiments, a polypeptide or agent comprises a polypeptide consisting of SEQ ID NO:97. In certain embodiments, a polypeptide or agent comprises at least a first, second, and third copy of a fragment of the extracellular domain of CD40L. In some embodiments, the copies of the extracellular domain of CD40L consist of the same amino acid sequence. In some embodiments, the copies of the extracellular domain of CD40L are not identical. In some embodiments, the copies of the extracellular domain of CD40L comprise substitutions, deletions, and/or additions to the amino acid sequence of human CD40L as compared to the wild-type sequence.

In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof, wherein at least one of the extracellular domains comprises the stalk region or a stalk region fragment. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof, wherein each extracellular domain comprises the stalk region or a stalk region fragment. In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, a polypeptide or agent is a single chain fusion polypeptide comprising at least a first, second, and third copy of the extracellular domain of human CD40L, wherein each extracellular domain comprises the stalk region or a stalk region fragment and the polypeptide does not comprise any peptide linkers (i.e., exogenous peptide linkers). In some embodiments, the extracellular domain of CD40L comprises amino acids 113-261 of SEQ ID NO:82. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:84. In some embodiments, the extracellular domain of OX40L consists of SEQ ID NO:84. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:103. In some embodiments, the extracellular domain of OX40L consists of SEQ ID NO:103. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:104. In some embodiments, the extracellular domain of OX40L consists of SEQ ID NO:104. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:105. In some embodiments, the extracellular domain of OX40L consists of SEQ ID NO:105. In some embodiments, the extracellular domain of CD40L comprises SEQ ID NO:106. In some embodiments, the extracellular domain of OX40L consists of SEQ ID NO:106. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:85. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:85. In some embodiments, the single chain fusion polypeptide comprises SEQ ID NO:97. In some embodiments, the single chain fusion polypeptide consists of SEQ ID NO:97.

In certain embodiments, a polypeptide or agent comprises a variant of the extracellular domain GITRL amino acid sequence or a fragment thereof that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding GITR. In certain embodiments, a polypeptide or agent comprises a variant of the extracellular domain OX40L amino acid sequence or a fragment thereof that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding OX40. In certain embodiments, a polypeptide or agent comprises a variant of the extracellular domain CD40L amino acid sequence or a fragment thereof that comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) conservative substitutions and is capable of binding CD40.

In some embodiments, the agent is a polypeptide. In some embodiments, the polypeptide is a fusion protein. In certain embodiments, the fusion protein comprises at least one copy of the extracellular domain of a member of the human TNFSF or a fragment thereof, and further comprises a non-TNFSF polypeptide. In certain embodiments, the fusion protein comprises at least one copy of the extracellular domain of human GITRL or a fragment thereof, and further comprises a non-GITRL polypeptide. In certain embodiments, the fusion protein comprises at least one copy of the extracellular domain of human OX40L or a fragment thereof, and further comprises a non-OX40L polypeptide. In certain embodiments, the fusion protein comprises at least one copy of the extracellular domain of human CD40L or a fragment thereof, and further comprises a non-CD40L polypeptide. In some embodiments, the fusion protein may include an extracellular domain or fragment thereof linked to a heterologous functional and structural polypeptide including, but not limited to, a human Fc region, one or more protein tags (e.g., myc, FLAG, GST), other endogenous proteins or protein fragments, or any other useful protein sequence including any peptide sequence between the extracellular domain and the non-TNFSF polypeptide (e.g., a non-GITRL polypeptide or a non-OX40L polypeptide). In certain embodiments, the non-TNFSF polypeptide comprises a human Fc region. In certain embodiments, the non-GITRL polypeptide comprises a human Fc region. In certain embodiments, the non-OX40L polypeptide comprises a human Fc region. In certain embodiments, the non-CD40L polypeptide comprises a human Fc region. The Fc region can be obtained from any of the classes of immunoglobulin, IgG, IgA, IgM, IgD and IgE. In some embodiments, the Fc region is a human IgG1 Fc region. In some embodiments, the Fc region is a human IgG2 Fc region. In some embodiments, the Fc region is a wild-type Fc region. In some embodiments, the Fc region is a natural variant of a wild-type Fc region. In some embodiments, the Fc region is a mutated Fc region. In some embodiments, the Fc region is truncated at the N-terminal end by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, (e.g., in the hinge domain). In some embodiments, the Fc region is truncated at the C-terminal end (e.g., lysine is absent). In some embodiments, an amino acid in the hinge domain is changed to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a different amino acid to hinder undesirable disulfide bond formation. In some embodiments, a cysteine is replaced with a serine to hinder undesirable disulfide bond formation. In some embodiments, the Fc region comprises SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In some embodiments, the Fc region comprise SEQ ID NO:10. In some embodiments, the Fc region comprises SEQ ID NO:14.

In some embodiments, the polypeptide or agent is a single chain GITRL trimer-Fc protein. In some embodiments, the polypeptide or agent is a single chain GITRL trimer-IgG1 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the polypeptide or agent comprises SEQ ID NO:6. In some embodiments, the polypeptide or agent comprises SEQ ID NO:7. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:6 or SEQ ID NO:7. In some embodiments, the polypeptide or agent consists of SEQ ID NO:6. In some embodiments, the polypeptide or agent consists of SEQ ID NO:7. In some embodiments, the polypeptide or agent comprises a polypeptide encoded by the “hGITRL-hIgG1” plasmid deposited with ATCC and assigned designation number PTA-122112. In some embodiments, the polypeptide or agent is a single chain GITRL trimer-IgG2 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the polypeptide or agent comprises SEQ ID NO:8. In some embodiments, the polypeptide or agent comprises SEQ ID NO:9. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:8 or SEQ ID NO:9. In some embodiments, the polypeptide or agent consists of SEQ ID NO:8. In some embodiments, the polypeptide or agent consists of SEQ ID NO:9.

In some embodiments, the polypeptide or agent is a single chain OX40L trimer-Fc protein. In some embodiments, the polypeptide or agent is a single chain OX40L trimer-IgG1 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:45 or SEQ ID NO:46. In some embodiments, the polypeptide or agent comprises SEQ ID NO:45. In some embodiments, the polypeptide or agent comprises SEQ ID NO:46. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:45 or SEQ ID NO:46. In some embodiments, the polypeptide or agent consists of SEQ ID NO:45. In some embodiments, the polypeptide or agent consists of SEQ ID NO:46. In some embodiments, the polypeptide or agent is a single chain OX40L trimer-IgG2 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:47 or SEQ ID NO:48. In some embodiments, the polypeptide or agent comprises SEQ ID NO:47. In some embodiments, the polypeptide or agent comprises SEQ ID NO:48. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:47 or SEQ ID NO:48. In some embodiments, the polypeptide or agent consists of SEQ ID NO:47. In some embodiments, the polypeptide or agent consists of SEQ ID NO:48.

In some alternative embodiments, the polypeptide or agent is a single chain OX40L trimer-IgG1 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:80 or SEQ ID NO:81. In some embodiments, the polypeptide or agent comprises SEQ ID NO:80. In some embodiments, the polypeptide or agent comprises SEQ ID NO:81. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:80 or SEQ ID NO:81. In some embodiments, the polypeptide or agent consists of SEQ ID NO:80. In some embodiments, the polypeptide or agent consists of SEQ ID NO:81.

In some embodiments, the polypeptide or agent is a single chain CD40L trimer-Fc protein. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG1 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:89 or SEQ ID NO:90. In some embodiments, the polypeptide or agent comprises SEQ ID NO:89. In some embodiments, the polypeptide or agent comprises SEQ ID NO:90. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:89 or SEQ ID NO:90. In some embodiments, the polypeptide or agent consists of SEQ ID NO:89. In some embodiments, the polypeptide or agent consists of SEQ ID NO:90. In some embodiments, the polypeptide or agent is a single chain CD40L trimer-IgG2 Fc protein. In some embodiments, the polypeptide or agent comprises SEQ ID NO:91 or SEQ ID NO:92. In some embodiments, the polypeptide or agent comprises SEQ ID NO:91. In some embodiments, the polypeptide or agent comprises SEQ ID NO:92. In some embodiments, the polypeptide or agent consists essentially of SEQ ID NO:91 or SEQ ID NO:92. In some embodiments, the polypeptide or agent consists of SEQ ID NO:91. In some embodiments, the polypeptide or agent consists of SEQ ID NO:92.

In certain embodiments, the non-TNFSF polypeptide (e.g., non-GITRL polypeptide, non-OX40L polypeptide, or non-CD40L polypeptide) comprises SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In certain embodiments, the non-TNFSF polypeptide (e.g., non-GITRL polypeptide, non-OX40L polypeptide, or non-CD40L polypeptide) consists essentially of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In certain embodiments, the non-TNFSF polypeptide (e.g., non-GITRL polypeptide, non-OX40L polypeptide, or non-CD40L polypeptide) consists of SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14.

In certain embodiments, the non-GITRL polypeptide or the non-OX40L polypeptide comprises an immunoglobulin heavy chain. In certain embodiments, the non-CD40L polypeptide comprises an immunoglobulin heavy chain. In certain embodiments, the immunoglobulin heavy chain is associated with an immunoglobulin light chain. In some embodiments, the immunoglobulin heavy chain and the immunoglobulin light chain form an antigen-binding site. In certain embodiments, the non-GITRL polypeptide or the non-OX40L polypeptide comprises an antibody. In certain embodiments, the non-CD40L polypeptide comprises an antibody. In certain embodiments, the non-GITRL polypeptide or the non-OX40L polypeptide comprises a single chain antibody or Fab. In certain embodiments, the non-CD40L polypeptide comprises a single chain antibody or Fab.

In certain embodiments, a fusion protein comprises a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof and a non-GITRL polypeptide, wherein the C-terminal end of the non-GITRL polypeptide is linked to the extracellular domain(s) of GITRL. In certain embodiments, a fusion protein comprises a first, second, and third copy of the extracellular domain of human GITRL or a fragment thereof and a non-GITRL polypeptide, wherein the N-terminal end of the non-GITRL polypeptide is linked to the extracellular domain(s) of GITRL. In some embodiments, the first copy of the extracellular domain of GITRL is linked to the C-terminal end of the non-GITRL polypeptide. In some embodiments, the third copy of the extracellular domain of GITRL is linked to the N-terminal end of the non-GITRL polypeptide. In some embodiments, the extracellular domain(s) of GITRL is linked to the C-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of GITRL is linked to the N-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of GITRL is directly linked to the Fc region (i.e. without an intervening peptide linker). In some embodiments, the extracellular domain(s) of GITRL is linked to the Fc region via a peptide linker.

In certain embodiments, a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human OX40L or a fragment thereof and a non-OX40L polypeptide, wherein the C-terminal end of the non-OX40L polypeptide is linked to the extracellular domain(s) of OX40L. In certain embodiments, a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human OX40L or a fragment thereof and a non-OX40L polypeptide, wherein the N-terminal end of the non-OX40L polypeptide is linked to the extracellular domain(s) of OX40L. In some embodiments, the first copy of the extracellular domain of OX40L is linked to the C-terminal end of the non-OX40L polypeptide. In some embodiments, the third copy of the extracellular domain of OX40L is linked to the N-terminal end of the non-OX40L polypeptide. In some embodiments, the extracellular domain(s) of OX40L is linked to the C-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of OX40L is linked to the N-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of OX40L is directly linked to the Fc region (i.e. without an intervening peptide linker). In some embodiments, the extracellular domain(s) of OX40L is linked to the Fc region via a peptide linker.

In certain embodiments, a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof and a non-CD40L polypeptide, wherein the C-terminal end of the non-CD40L polypeptide is linked to the extracellular domain(s) of CD40L. In certain embodiments, a fusion protein comprises at least a first, second, and third copy of the extracellular domain of human CD40L or a fragment thereof and a non-CD40L polypeptide, wherein the N-terminal end of the non-CD40L polypeptide is linked to the extracellular domain(s) of CD40L. In some embodiments, the first copy of the extracellular domain of CD40L is linked to the C-terminal end of the non-CD40L polypeptide. In some embodiments, the third copy of the extracellular domain of CD40L is linked to the N-terminal end of the non-CD40L polypeptide. In some embodiments, the extracellular domain(s) of CD40L is linked to the C-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of CD40L is linked to the N-terminal end of a Fc region. In some embodiments, the extracellular domain(s) of CD40L is directly linked to the Fc region (i.e. without an intervening peptide linker). In some embodiments, the extracellular domain(s) of CD40L is linked to the Fc region via a peptide linker.

As used herein, the term “linker” refers to a linker inserted between a first polypeptide (e.g., a extracellular domain of TNFSF (e.g., GITRL, OX40L, or CD40L or a fragment thereof) and a second polypeptide (e.g., a Fc region). In some embodiments, the linker is a peptide linker. Linkers should not adversely affect the expression, secretion, or bioactivity of the fusion protein. Linkers should not be antigenic and should not elicit an immune response. Suitable linkers are known to those of skill in the art and often include mixtures of glycine and serine residues and often include amino acids that are sterically unhindered. Other amino acids that can be incorporated into useful linkers include threonine and alanine residues. Linkers can range in length, for example from 1-50 amino acids in length, 1-22 amino acids in length, 1-10 amino acids in length, 1-5 amino acids in length, or 1-3 amino acids in length. Linkers may include, but are not limited to, SerGly, GGSG, GSGS, GGGS, S(GGS)n where n is 1-7, GRA, poly(Gly), poly(Ala), GGGSGGG (SEQ ID NO:57), ESGGGGVT (SEQ ID NO:34), LESGGGGVT (SEQ ID NO:35), GRAQVT (SEQ ID NO:36), WRAQVT (SEQ ID NO:37), and ARGRAQVT (SEQ ID NO:38). In some embodiments, the linker may comprise a cleavage site. In some embodiments, the linker may comprise an enzyme cleavage site, so that the second polypeptide may be separated from the first polypeptide. As used herein, a linker is an intervening peptide sequence that does not include amino acid residues from either the C-terminus of the first polypeptide (e.g., an extracellular domain of GITRL, OX40L, or CD40L) or the N-terminus of the second polypeptide (e.g., the Fc region).

In some embodiments, a polypeptide or agent described herein specifically binds GITR and acts as a GITR agonist. In some embodiments, a polypeptide or agent described herein specifically binds GITR and activates GITR signaling. In some embodiments, a polypeptide or agent described herein specifically binds GITR and induces, activates, promotes, increases, enhances, or prolongs GITR activity. In some embodiments, a polypeptide or agent described herein specifically binds OX40 and acts as an OX40 agonist. In some embodiments, a polypeptide or agent described herein specifically binds OX40 and activates OX40 signaling. In some embodiments, a polypeptide or agent described herein specifically binds OX40 and induces, activates, promotes, increases, enhances, or prolongs OX40 activity. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and acts as an CD40 agonist. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and activates CD40 signaling. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and induces, activates, promotes, increases, enhances, or prolongs CD40 activity.

In some embodiments, a polypeptide or agent described herein specifically binds GITR and modulates an immune response. In some embodiments, a polypeptide or agent described herein specifically binds GITR and induces, augments, increases, and/or prolongs an immune response. In some embodiments, a polypeptide or agent described herein specifically binds OX40 and modulates an immune response. In some embodiments, a polypeptide or agent described herein specifically binds OX40 and induces, augments, increases, and/or prolongs an immune response. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and modulates an immune response. In some embodiments, a polypeptide or agent described herein specifically binds CD40 and induces, augments, increases, and/or prolongs an immune response.

In some embodiments, a polypeptide or agent described herein specifically binds a member of the TNFRSF (e.g., GITR, OX40, or CD40) with a dissociation constant (K_(D)) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less. In some embodiments, a polypeptide or agent binds a member of the TNFRSF (e.g., GITR, OX40, or CD40) with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds a member of the TNFRSF (e.g., GITR, OX40, or CD40) with a K_(D) of about 0.1 nM or less. In some embodiments, a polypeptide or agent binds human TNFRSF and/or mouse TNFRSF with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human TNFRSF with a K_(D) of about 10 nM or less.

In some embodiments, a polypeptide or agent binds human GITR and/or mouse GITR with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human GITR and/or mouse GITR with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds human GITR and/or mouse GITR with a K_(D) of about 0.1 nM or less. In some embodiments, a polypeptide or agent binds human GITR and does not bind mouse GITR. In some embodiments, a polypeptide or agent binds human GITR with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human GITR with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds human GITR with a K_(D) of about 0.1 nM or less.

In some embodiments, a polypeptide or agent binds human OX40 and/or mouse OX40 with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human OX40 and/or mouse OX40 with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds human OX40 and/or mouse OX40 with a K_(D) of about 0.1 nM or less. In some embodiments, a polypeptide or agent binds human OX40 and does not bind mouse OX40. In some embodiments, a polypeptide or agent binds human OX40 with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human OX40 with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds human OX40 with a K_(D) of about 0.1 nM or less.

In some embodiments, a polypeptide or agent binds human CD40 and/or mouse CD40 with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human CD40 and/or mouse CD40 with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds human CD40 and/or mouse CD40 with a K_(D) of about 0.1 nM or less. In some embodiments, a polypeptide or agent binds human CD40 and does not bind mouse CD40. In some embodiments, a polypeptide or agent binds human CD40 with a K_(D) of about 10 nM or less. In some embodiments, a polypeptide or agent binds human CD40 with a K_(D) of about 1 nM or less. In some embodiments, a polypeptide or agent binds human CD40 with a K_(D) of about 0.1 nM or less.

In some embodiments, the dissociation constant of the polypeptide or agent to a member of the TNFRSF (e.g., GITR, OX40, or CD40) is the dissociation constant determined using a TNFRSF fusion protein comprising at least a portion of a TNFRSF extracellular domain immobilized on a Biacore chip.

In some embodiments, a polypeptide or agent binds a member of the TNFRSF (e.g., human GITR, OX40, or CD40) with a half maximal effective concentration (EC₅₀) of about 1 μM or less, about 100 nM or less, about 40 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, or about 0.1 nM or less.

In certain embodiments, fusion polypeptides are made using recombinant DNA techniques as known to one skilled in the art. In some embodiments, polynucleotides encoding a specific protein or a fragment thereof are isolated from mammalian cells, such as by RT-PCR using oligonucleotide primers that specifically amplify the gene encoding the protein, and the nucleotide sequence is determined using conventional techniques. The isolated polynucleotides encoding the protein may be cloned into suitable expression vectors which produce the polypeptide when transfected into host cells such as E. coli, simian COS cells, or Chinese hamster ovary (CHO) cells. In other embodiments, recombinant proteins, or fragments thereof, can be isolated from phage display libraries or using other cell surface display techniques.

The polynucleotide(s) encoding a protein can be modified in a number of different manners using recombinant DNA technology to generate alternative or variant proteins. Site-directed or high-density mutagenesis of a protein can be used to optimize specificity, affinity, stability, etc. of a recombinant protein.

Proteins generally contain a signal sequence that directs the transport of the proteins. Signal sequences (also referred to as signal peptides or leader sequences) are located at the N-terminus of nascent polypeptides. They target the polypeptide to the endoplasmic reticulum and the proteins are sorted to their destinations, for example, to the inner space of an organelle, to an interior membrane, to the cell outer membrane, or to the cell exterior via secretion. Most signal sequences are cleaved from the protein by a signal peptidase after the proteins are transported to the endoplasmic reticulum. The cleavage of the signal sequence from the polypeptide usually occurs at a specific site in the amino acid sequence and is dependent upon amino acid residues within the signal sequence. Although there is usually one specific cleavage site, more than one cleavage site may be recognized and/or used by a signal peptidase resulting in a non-homogenous N-terminus of the polypeptide. For example, the use of different cleavage sites within a signal sequence can result in a polypeptide expressed with different N-terminal amino acids. Accordingly, in some embodiments, the polypeptides as described herein may comprise a mixture of polypeptides with different N-termini. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids. In some embodiments, the N-termini differ in length by 1, 2, 3, 4, or 5 amino acids. In some embodiments, the polypeptide is substantially homogeneous, i.e., the polypeptides have the same N-terminus. In some embodiments, the signal sequence of the polypeptide comprises one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, etc.) amino acid substitutions and/or deletions as compared to the native sequence of the protein. In some embodiments, the signal sequence of the polypeptide comprises amino acid substitutions and/or deletions that allow one cleavage site to be dominant, thereby resulting in a substantially homogeneous polypeptide with one N-terminus. In some embodiments, the signal sequence of a fusion polypeptide is not the native signal sequence of the protein(s) contained within the fusion polypeptide.

In certain embodiments, a polypeptide, agent, or fusion polypeptide described herein comprises the Fc region of an immunoglobulin Those skilled in the art will appreciate that some of the polypeptides or agents of this invention will comprise fusion proteins in which at least a portion of the Fc region has been deleted or otherwise altered so as to provide desired biochemical characteristics, such as increased cancer cell localization, increased tumor penetration, reduced serum half-life, or increased serum half-life, when compared with a fusion protein of approximately the same immunogenicity comprising a native or unaltered Fc region. Modifications to the Fc region may include additions, deletions, or substitutions of one or more amino acids in one or more domains. The modified fusion proteins disclosed herein may comprise alterations or modifications to one or more of the two heavy chain constant domains (CH2 or CH3) or to the hinge region. In other embodiments, the entire CH2 domain may be removed (ΔCH2 constructs). In some embodiments, the omitted constant region domain is replaced by a short amino acid spacer (e.g., 10 aa residues) that provides some of the molecular flexibility typically imparted by the absent constant region domain.

In some embodiments, the modified fusion proteins are engineered to link the CH3 domain directly to the hinge region or to the first polypeptide. In other embodiments, a peptide spacer or linker is inserted between the hinge region or the first polypeptide and the modified CH2 and/or CH3 domains. For example, constructs may be expressed wherein the CH2 domain has been deleted and the remaining CH3 domain (modified or unmodified) is joined to the hinge region or first polypeptide with a 5-20 amino acid spacer. Such a spacer may be added to ensure that the regulatory elements of the constant domain remain free and accessible or that the hinge region remains flexible. However, it should be noted that amino acid spacers may, in some cases, prove to be immunogenic and elicit an unwanted immune response against the construct. Accordingly, in certain embodiments, any spacer added to the construct will be relatively non-immunogenic so as to maintain the desired biological qualities of the fusion protein.

In some embodiments, the modified fusion proteins may have only a partial deletion of a constant domain or substitution of a few or even a single amino acid. For example, the mutation of a single amino acid in selected areas of the CH2 domain may be enough to substantially reduce Fc binding and thereby increase cancer cell localization and/or tumor penetration. Similarly, it may be desirable to simply delete that part of one or more constant region domains that control a specific effector function (e.g., complement Clq binding). Such partial deletions of the constant regions may improve selected characteristics of the polypeptide or agent (e.g., serum half-life) while leaving other desirable functions associated with the subject constant region domain intact. Moreover, as alluded to above, the constant regions of the disclosed fusion proteins may be modified through the mutation or substitution of one or more amino acids that enhances the profile of the resulting construct. In this respect it may be possible to disrupt the activity provided by a conserved binding site (e.g., Fc binding) while substantially maintaining the configuration and immunogenic profile of the modified fusion protein. In certain embodiments, the modified fusion proteins comprise the addition of one or more amino acids to the constant region to enhance desirable characteristics such as decreasing or increasing effector function, or provide for more cytotoxin or carbohydrate attachment sites.

It is known in the art that the constant region mediates several effector functions. For example, binding of the C1 component of complement to the Fc region of IgG or IgM antibodies (bound to antigen) activates the complement system. Activation of complement is important in the opsonization and lysis of cell pathogens. The activation of complement also stimulates the inflammatory response and can also be involved in autoimmune hypersensitivity. In addition, the Fc region can bind to a cell expressing a Fc receptor (FcR). There are a number of Fc receptors which are specific for different classes of antibody, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors).

In some embodiments, the modified fusion proteins provide for altered effector functions that, in turn, affect the biological profile of the polypeptide or agent. For example, in some embodiments, the deletion or inactivation (through point mutations or other means) of a constant region domain may reduce Fc receptor binding of the circulating modified agent, thereby increasing cancer cell localization and/or tumor penetration. In other embodiments, the constant region modifications increase or reduce the serum half-life of the polypeptide or agent. In some embodiments, the constant region is modified to eliminate disulfide linkages or oligosaccharide moiety attachment sites.

In certain embodiments, a modified fusion protein does not have one or more effector functions normally associated with an Fc region. In some embodiments, the polypeptide or agent has no antibody-dependent cell-mediated cytotoxicity (ADCC) activity, and/or no complement-dependent cytotoxicity (CDC) activity. In certain embodiments, the polypeptide or agent does not bind to the Fc receptor and/or complement factors. In certain embodiments, the polypeptide or agent has no effector function normally associated with an Fc region.

The polypeptides and agents of the present invention can be assayed for specific binding to a target by any method known in the art. The immunoassays which can be used include, but are not limited to, competitive and non-competitive assay systems using techniques such as Biacore analyses, FACS analyses, immunofluorescence, immunocytochemistry, Western blot analyses, radioimmunoassays, ELISAs, “sandwich” immunoassays, immunoprecipitation assays, precipitation reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, and protein A immunoassays. Such assays are routine and well-known in the art.

For example, the specific binding of a test agent (e.g., a polypeptide) to human GITR may be determined using ELISA. An ELISA assay comprises preparing GITR protein, coating wells of a 96-well microtiter plate with the GITR, adding the test agent conjugated to a detectable compound such as an enzymatic substrate (e.g. horseradish peroxidase or alkaline phosphatase) to the well, incubating for a period of time and detecting the presence of the agent bound to GITR. In some embodiments, the test agent is not conjugated to a detectable compound, but instead a labeled secondary antibody that recognizes the agent is added to the well. In some embodiments, instead of coating the well with GITR, the test agent can be coated to the well, GITR is added, and a second antibody conjugated to a detectable compound that recognizes GITR can be used to detect binding. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art.

In another example, the specific binding of a test agent e.g., a polypeptide) to human GITR may be determined using FACS. A FACS screening assay may comprise generating a cDNA construct that expresses GITR, transfecting the construct into cells, expressing GITR on the surface of the cells, mixing the test agent with the transfected cells, and incubating for a period of time. The cells bound by the test agent may be identified by using a secondary antibody conjugated to a detectable compound (e.g., PE-conjugated anti-Fc antibody) and a flow cytometer. One of skill in the art would be knowledgeable as to the parameters that can be modified to optimize the signal detected as well as other variations of FACS that may enhance screening (e.g., screening for blocking antibodies).

The binding affinity of a test agent to a target (e.g., human GITR) and the off-rate of an agent-target interaction can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay comprising the incubation of labeled target (e.g., ³H or ¹²⁵I-labeled GITR), or fragment or variant thereof, with the agent of interest in the presence of increasing amounts of unlabeled target followed by the detection of the agent bound to the labeled target. The affinity of the agent for a target (e.g., human GITR) and the binding off-rates can be determined from the data by Scatchard plot analysis. In some embodiments, Biacore kinetic analysis is used to determine the binding on and off rates of agents that bind a target (e.g., human GITR). Biacore kinetic analysis comprises analyzing the binding and dissociation of agents from chips with immobilized target (e.g., human GITR) on the chip surface.

This invention also encompasses homodimeric agents and heterodimeric agents/molecules. In some embodiments, the homodimeric agents are polypeptides. In some embodiments, the heterodimeric molecules are polypeptides. Generally the homodimeric molecule comprises two identical polypeptides. Generally the heterodimeric molecule comprises two non-identical polypeptides. In some embodiments, a heterodimeric molecule is capable of binding at least two targets, e.g., a bispecific agent. The targets may be, for example, two different proteins on a single cell or two different proteins on two separate cells. In some embodiments, the bispecific agents are polypeptides. Thus, in some embodiments, one polypeptide of the heterodimeric molecule comprises a polypeptide described herein (e.g., a single chain trimer-Fc protein that binds GITR, OX40, or CD40) and one polypeptide of the heterodimeric molecule is an antibody. The term “arm” may be used herein to describe the structure of a homodimeric agent, a heterodimeric agent, and/or a bispecific agent. As used herein, each “arm” is directed against a target. In some embodiments, one “arm” may comprise an antigen-binding site from an antibody. In some embodiments, one “arm” may comprise a binding portion of a receptor. In some embodiments, a homodimeric agent comprises two identical arms. In some embodiments, a heterodimeric agent comprises two different arms. In some embodiments, a bispecific agent comprises two different arms.

In some embodiments, a bispecific agent comprises the polypeptides or agents described herein. In some embodiments, the bispecific agent is a homodimeric protein (FIG. 3(ii) shows representative depictions). In some embodiments, the homodimer bispecific agent comprises a polypeptide comprising a heavy chain immunoglobulin and a TNFSF trimer. In some embodiments, the heavy chain immunoglobulin is associated with a light chain to form an antigen-binding site. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain TNFSF trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising a single-chain antibody and a single chain TNFSF trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain GITRL trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain OX40L trimer. In some embodiments, the homodimeric bispecific agent comprises a polypeptide comprising an antibody and a single chain CD40L trimer. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds a tumor antigen. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds an antigen on an immune cell. In some embodiments, the homodimeric bispecific agent comprises an antibody that specifically binds PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, or TIM3. In some embodiments, the homodimeric bispecific agent binds GITR and PD-1. In some embodiments, the homodimeric bispecific agent binds GITR and PD-L1. In some embodiments, the homodimeric bispecific agent binds OX-40 and PD-1. In some embodiments, the homodimeric bispecific agent binds OX-40 and PD-L1. In some embodiments, the homodimeric bispecific agent binds CD40 and PD-1. In some embodiments, the homodimeric bispecific agent binds CD40 and PD-L1.

In some embodiments, the bispecific agent is a heterodimeric protein (FIG. 3(iii) shows a representative depiction). In some embodiments, the heterodimeric bispecific agent comprises an antigen-binding site from an antibody (e.g., an antigen-binding site formed by an immunoglobulin heavy chain and an immunoglobulin light chain) and a TNFSF trimer. In certain embodiments, a bispecific agent comprises an immune response stimulating agent or functional fragment thereof and a TNFSF trimer.

In some embodiments, a heterodimeric bispecific agent is capable of binding one target and also comprises a “non-binding” function. Thus in some embodiments, one polypeptide of the heterodimeric bispecific agent comprises a polypeptide described herein (e.g., binds a TNFRSF such as GITR, OX40, or CD40) and one polypeptide of the heterodimeric agent is an additional immune response stimulating agent. As used herein, the phrase “immune response stimulating agent” is used in the broadest sense and refers to a substance that directly or indirectly stimulates the immune system by inducing activation or increasing activity of any of the immune system's components. For example, immune response stimulating agents may include cytokines, as well as various antigens including tumor antigens, and antigens derived from pathogens. In some embodiments, the immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28 antibody, anti-PD-1 antibody, anti-PD-L1 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86).

In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds a tumor antigen. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a OX40L trimer and a second polypeptide comprising an antibody that specifically binds a tumor antigen. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds a tumor antigen. A bispecific agent with a binding specificity for a tumor antigen can be used to direct the GITRL, OX40L, or CD40L trimer polypeptide to a tumor. For example the bispecific agent may be used to direct the GITRL, OX40L, or CD40L trimer polypeptide to a tumor that expresses the tumor antigen or overexpresses the tumor antigen. This may be useful to induce and/or enhance an immune response near or within the tumor microenvironment. In some embodiments, a bispecific agent may be used to induce or enhance the activity of tumor infiltrating immune cells.

In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a TNFSF trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a OX40L trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds an immune response molecule. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds an immune checkpoint protein. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising an OX40L trimer and a second polypeptide comprising an antibody that specifically binds an immune checkpoint protein. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds an immune checkpoint protein. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds PD-1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a GITRL trimer and a second polypeptide comprising an antibody that specifically binds PD-L1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising an OX40L trimer and a second polypeptide comprising an antibody that specifically binds PD-1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising an OX40L trimer and a second polypeptide comprising an antibody that specifically binds PD-L1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds PD-1. In some embodiments, a heterodimeric bispecific agent comprises a first polypeptide comprising a CD40L trimer and a second polypeptide comprising an antibody that specifically binds PD-L1.

In some embodiments, the heterodimeric molecule (e.g., a bispecific agent) can bind a first target, (e.g., GITR, OX40, or CD40) as well as a second target, such as an effector molecule on a leukocyte (e.g., CD2, CD3, CD28, or CD80) or a Fc receptor (e.g., CD64, CD32, or CD16) so as to elicit a stronger cellular immune response.

In some embodiments, a bispecific agent, either heterodimeric or homodimeric, has enhanced potency as compared to an individual agent. It is known to those of skill in the art that any agent (e.g., a soluble protein or a cytokine) may have unique pharmacokinetics (PK) (e.g., circulating half-life). In some embodiments, a bispecific agent has the ability to synchronize the PK of two active agents and/or polypeptides wherein the two individual agents and/or polypeptides have different PK profiles. In some embodiments, a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides in a common area (e.g., a tumor and/or tumor microenvironment). In some embodiments, a bispecific molecule has the ability to concentrate the actions of two agents and/or polypeptides to a common target (e.g., a tumor or a tumor cell). In some embodiments, a bispecific agent has the ability to target the actions of two agents and/or polypeptides to more than one biological pathway or more than one aspect of the immune response. In some embodiments, the bispecific agent has decreased toxicity and/or side effects than either of the polypeptides and/or agents alone. In some embodiments, the bispecific agent has decreased toxicity and/or side effects as compared to a mixture of the two individual polypeptides and/or agents. In some embodiments, the bispecific agent has an increased therapeutic index. In some embodiments, the bispecific agent has an increased therapeutic index as compared to a mixture of the two individual polypeptides and/or agents or the polypeptides and/or agents as single agents.

It is believed that a single chain TNFSF molecule may be more active than an anti-TNFSF agonist antibody, because a single chain TNFSF trimer would function, i.e., bind to three TNFRSF molecules, in a very similar manner as three native TNFSF molecules. In contrast, an anti-TNFRSF antibody is able to engage only two TNFRSF molecules, thereby reducing any potential effect. To make a bispecific molecule comprising an antibody, generally involves the antibody being monovalent (i.e., one-armed antibody). This reduces, if not completely eliminates, the effect of an agonist antibody, especially if activation depends upon clustering of the target molecules. A single chain TNFSF (e.g., a GITRL trimer, an OX40L trimer, or a CD40L trimer) is able to bind three TNFRSF molecules, thus as part of a heterodimeric or homodimeric bispecific molecule, it does not lose any functionality or potency.

In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain TNFSF trimer and a second polypeptide comprising an antibody. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain TNFSF trimer and a second polypeptide comprising an antagonist antibody. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain GITRL trimer and a second polypeptide comprising an antagonist antibody. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain OX40L trimer and a second polypeptide comprising an antagonist antibody. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain CD40L trimer and a second polypeptide comprising an antagonist antibody.

In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of GITRL or a GITR-binding fragment thereof, and (b) a second arm comprising an antigen-binding site from an antibody. In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of GITRL or a GITR-binding fragment thereof, and (b) a second arm comprising an immune response stimulating agent. In some embodiments, at least one copy of the extracellular domain of GITRL of the first arm comprises SEQ ID NO:3 or SEQ ID NO:64. In some embodiments, the heterodimeric bispecific agent comprises a first arm comprising SEQ ID NO:5 or SEQ ID NO:66. In some embodiments, the heterodimeric bispecific agent comprises a first arm which further comprises a non-GITRL polypeptide. In some embodiments, the heterodimeric bispecific agent comprises a single chain fusion GITRL polypeptide described herein which is directly linked to a non-GITRL polypeptide. In some embodiments, the single chain fusion polypeptide is connected to the non-GITRL polypeptide by a linker. In some embodiments, the non-GITRL polypeptide comprises a human Fc region. In some embodiments, the non-GITRL polypeptide comprises SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61.

In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of OX40L or an OX40-binding fragment thereof, and (b) a second arm comprising an antigen-binding site from an antibody. In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of OX40L or an OX40-binding fragment thereof, and (b) a second arm comprising an immune response stimulating agent. In some embodiments, at least one copy of the extracellular domain of OX40L of the first arm comprises SEQ ID NO:42, SEQ ID NO:67, SEQ ID NO:78, or SEQ ID NO:79. In some embodiments, the bispecific agent comprises a first arm comprising SEQ ID NO:44, SEQ ID NO:69, SEQ ID NO:71, or SEQ ID NO:72. In some embodiments, the heterodimeric bispecific agent comprises a first arm which further comprises a non-OX40L polypeptide. In some embodiments, the heterodimeric bispecific agent comprises a single chain fusion OX40L polypeptide described herein which is directly linked to a non-OX40L polypeptide. In some embodiments, the single chain fusion polypeptide is connected to the non-OX40L polypeptide by a linker. In some embodiments, the non-OX40L polypeptide comprises a human Fc region. In some embodiments, the non-OX40L polypeptide comprises SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61.

In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of CD40L or an CD40-binding fragment thereof, and (b) a second arm comprising an antigen-binding site from an antibody. In some embodiments, a heterodimeric bispecific agent comprises: (a) a first arm comprising a single chain fusion polypeptide comprising a first, second, and third copy of the extracellular domain of CD40L or an CD40-binding fragment thereof, and (b) a second arm comprising an immune response stimulating agent. In some embodiments, at least one copy of the extracellular domain of CD40L of the first arm comprises SEQ ID NO:84, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, or SEQ ID NO:106. In some embodiments, the bispecific agent comprises a first arm comprising SEQ ID NO:85 or SEQ ID NO:97. In some embodiments, the heterodimeric bispecific agent comprises a first arm which further comprises a non-CD40L polypeptide. In some embodiments, the heterodimeric bispecific agent comprises a single chain fusion CD40L polypeptide described herein which is directly linked to a non-CD40L polypeptide. In some embodiments, the single chain fusion polypeptide is connected to the non-CD40L polypeptide by a linker. In some embodiments, the non-CD40L polypeptide comprises a human Fc region. In some embodiments, the non-CD40L polypeptide comprises SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, or SEQ ID NO:61.

In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain TNFSF trimer and a second polypeptide comprising an immune response stimulating agent. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain GITRL trimer and a second polypeptide comprising an immune response stimulating agent. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain OX40 trimer and a second polypeptide comprising an immune response stimulating agent. In some embodiments, a heterodimeric bispecific molecule comprises a first polypeptide comprising a single chain CD40 trimer and a second polypeptide comprising an immune response stimulating agent.

In some embodiments, the multimeric molecule (e.g., a bispecific agent) comprises a first CH3 domain and a second CH3 domain, each of which is modified to promote formation of heteromultimers or heterodimers. In some embodiments, the first and second CH3 domains are modified using a knobs-into-holes technique. In some embodiments, the first and second CH3 domains comprise changes in amino acids that result in altered electrostatic interactions. In some embodiments, the first and second CH3 domains comprise changes in amino acids that result in altered hydrophobic/hydrophilic interactions (see, for example, U.S. Patent App. Publication No. 2011/0123532).

In some embodiments, the heterodimeric molecule (e.g., a bispecific agent) comprises heavy chain constant regions selected from the group consisting of: (a) a first human IgG1 constant region, wherein the amino acids at positions corresponding to positions 253 and 292 of SEQ ID NO:15 are replaced with glutamate or aspartate, and a second human IgG1 constant region, wherein the amino acids at positions corresponding to 240 and 282 of SEQ ID NO:15 are replaced with lysine; (b) a first human IgG2 constant region, wherein the amino acids at positions corresponding to positions 249 and 288 of SEQ ID NO:16 are replaced with glutamate or aspartate, and a second human IgG2 constant region wherein the amino acids at positions corresponding to positions 236 and 278 of SEQ ID NO:16 are replaced with lysine; (c) a first human IgG3 constant region, wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:17 are replaced with glutamate or aspartate, and a second human IgG3 constant region wherein the amino acids at positions corresponding to positions 287 and 329 of SEQ ID NO:17 are replaced with lysine; and (d) a first human IgG4 constant region, wherein the amino acids at positions corresponding to positions 250 and 289 of SEQ ID NO:18 are replaced with glutamate or aspartate, and a second IgG4 constant region wherein the amino acids at positions corresponding to positions 237 and 279 of SEQ ID NO:18 are replaced with lysine.

In some embodiments, the heterodimeric molecule (e.g., a bispecific agent) comprises heavy chain CH2 and CH3 domains selected from the group consisting of: (a) a first human IgG1 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 253 and 292 of SEQ ID NO:15 are replaced with glutamate or aspartate, and a second human IgG1 CH2 and CH3 domain, wherein the amino acids at positions corresponding to 240 and 282 of SEQ ID NO:15 are replaced with lysine; (b) a first human IgG2 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 249 and 288 of SEQ ID NO:16 are replaced with glutamate or aspartate, and a second human IgG2 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 236 and 278 of SEQ ID NO:16 are replaced with lysine; (c) a first human IgG3 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 300 and 339 of SEQ ID NO:17 are replaced with glutamate or aspartate, and a second human IgG3 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 287 and 329 of SEQ ID NO:17 are replaced with lysine; and (d) a first human IgG4 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 250 and 289 of SEQ ID NO:18 are replaced with glutamate or aspartate, and a second IgG4 CH2 and CH3 domain, wherein the amino acids at positions corresponding to positions 237 and 279 of SEQ ID NO:18 are replaced with lysine.

In some embodiments, the heterodimeric molecule comprises two arms, wherein the first arm comprises a first human IgG1 constant region with amino acids substitutions at positions corresponding to positions 253 and 292 of SEQ ID NO:15, wherein the amino acids are replaced with glutamate or aspartate, and the second arm comprises a second human IgG1 constant region with amino acid substitutions at positions corresponding to positions 240 and 282 of SEQ ID NO:15, wherein the amino acids are replaced with lysine. In some embodiments, the two arms comprise a Fc region selected from the group consisting of SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, and SEQ ID NO:61. In some embodiments, the first arm comprises SEQ ID NO:58 and the second arm comprises SEQ ID NO:59. In some embodiments, the first arm comprises SEQ ID NO:60 and the second arm comprises SEQ ID NO:61.

In some embodiments, the heterodimeric molecule comprises two arms, wherein the first arm comprises a first human IgG2 constant region with amino acids substitutions at positions corresponding to positions 249 and 288 of SEQ ID NO:16, wherein the amino acids are replaced with glutamate or aspartate, and the second arm comprises a second human IgG2 constant region with amino acid substitutions at positions corresponding to positions 236 and 278 of SEQ ID NO:16, wherein the amino acids are replaced with lysine. In some embodiments, the two arms comprise a Fc region selected from the group consisting of SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31. In some embodiments, the first arm comprises SEQ ID NO:26 and the second arm comprises SEQ ID NO:27. In some embodiments, the first arm comprises SEQ ID NO:28 and the second arm comprises SEQ ID NO:30. In some embodiments, the first arm comprises SEQ ID NO:29 and the second arm comprises SEQ ID NO:31.

In some embodiments, the polypeptides or agents are monovalent. In some embodiments, the polypeptide or agent is a soluble protein that is monovalent. In some embodiments, the polypeptides or agents described herein are bivalent. In some embodiments, the polypeptides or agents described herein are trivalent. In some embodiments, the polypeptides or agents described herein are monospecific. In some embodiments, the polypeptides or agents described herein are bispecific. In some embodiments, the polypeptides or agents described herein are multispecific. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is monovalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is bivalent. In some embodiments, the agent is a heterodimeric protein that comprises two arms wherein at least one arm is trivalent (i.e., binds three target molecules).

In some embodiments, the polypeptides or agents comprise polypeptides that are substantially homologous to the fusion proteins and/or polypeptides described herein. These agents can contain, for example, conservative substitution mutations, i.e. the substitution of one or more amino acids by similar amino acids. For example, conservative substitution refers to the substitution of an amino acid with another within the same general class such as, for example, one acidic amino acid with another acidic amino acid, one basic amino acid with another basic amino acid, or one neutral amino acid by another neutral amino acid. What is intended by a conservative amino acid substitution is well known in the art and described herein.

In certain embodiments, a polypeptide or agent described herein binds GITR and modulates an immune response. In certain embodiments, a polypeptide or agent described herein binds OX40 and modulates an immune response. In certain embodiments, a polypeptide or agent described herein binds CD40 and modulates an immune response. In some embodiments, a polypeptide or agent described herein activates and/or increases an immune response. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances innate cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances adaptive cell-mediated immunity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CD4+ T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CD8+ T-cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances CTL activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances NK cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances lymphokine-activated killer cell (LAK) activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances tumor-infiltrating lymphocyte (TIL) activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases Treg cell activity. In some embodiments, a polypeptide or agent described herein inhibits or decreases MDSC cell activity. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances tumor cell killing. In some embodiments, a polypeptide or agent described herein increases, promotes, or enhances the inhibition of tumor growth. In some embodiments, a polypeptide or agent described herein increases or enhances an effective immune response without causing substantial side effects and/or immune-based toxicities. In some embodiments, a polypeptide or agent described herein increases or enhances an effective immune response without causing cytokine release syndrome (CRS) or a cytokine storm.

In some embodiments, a polypeptide or agent described herein binds GITR and induces, enhances, increases, and/or prolongs GITR signaling. In some embodiments, a polypeptide or agent described herein binds OX40 and induces, enhances, increases, and/or prolongs OX40 signaling. In some embodiments, a polypeptide or agent described herein binds CD40 and induces, enhances, increases, and/or prolongs CD40 signaling.

In certain embodiments, a polypeptide or agent described herein is an agonist (either directly or indirectly) of human GITR. In some embodiments, a polypeptide or agent is an agonist of GITR and activates and/or increases an immune response. In some embodiments, a polypeptide or agent is an agonist of GITR and activates and/or increases activity of NK cells and/or T-cells (e.g., cytolytic activity or cytokine production). In certain embodiments, a polypeptide or agent increases the activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%.

In certain embodiments, a polypeptide or agent described herein is an agonist (either directly or indirectly) of human OX40. In some embodiments, a polypeptide or agent is an agonist of OX40 and activates and/or increases an immune response. In some embodiments, a polypeptide or agent is an agonist of OX40 and activates and/or increases activity of NK cells and/or T-cells (e.g., cytolytic activity or cytokine production). In certain embodiments, a polypeptide or agent increases the activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%.

In certain embodiments, a polypeptide or agent described herein is an agonist (either directly or indirectly) of human CD40. In some embodiments, a polypeptide or agent is an agonist of CD40 and activates and/or increases an immune response. In some embodiments, a polypeptide or agent is an agonist of CD40 and activates and/or increases activity of NK cells and/or T-cells (e.g., cytolytic activity or cytokine production). In certain embodiments, a polypeptide or agent increases the activity by at least about 10%, at least about 20%, at least about 30%, at least about 50%, at least about 75%, at least about 90%, or about 100%.

In certain embodiments, a polypeptide or agent described herein increases activation of a NK cell. In certain embodiments, a polypeptide or agent increases activation of a T-cell. In certain embodiments, the activation of a NK cell and/or a T-cell by a polypeptide or agent results in an increase in the level of activation of a NK cell and/or a T-cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.

In certain embodiments, a polypeptide or agent described herein inhibits or decreases the suppressive activity of a Treg cell. In certain embodiments, a polypeptide or agent inhibits activity of a Treg cell. In certain embodiments, the inhibition of suppressive activity of a Treg cell by a polypeptide or agent results in an inhibition of suppressive activity of a Treg cell of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.

In certain embodiments, a polypeptide or agent described herein inhibits or decreases the suppressive activity of a MDSC. In certain embodiments, a polypeptide or agent inhibits activity of a MDSC. In certain embodiments, the inhibition of suppressive activity of a MDSC by a polypeptide or agent results in an inhibition of suppressive activity of a MDSC of at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, or at least about 95%.

In vivo and in vitro assays for determining whether a polypeptide or agent (or candidate binding agent) modulates an immune response are known in the art or are being developed. In some embodiments, a functional assay that detects T-cell activation can be used. In some embodiments, a functional assay that detects Treg activity can be used. In some embodiments, a functional assay that detects MDSC activity can be used. In some embodiments, a functional assay that detects NK cell activity can be used. In some embodiments, a functional assay that detects cytolytic T-cell activity can be used. In some embodiments, an assay that detects cytokine production can be used. In some embodiments, an assay that detects cytokine-producing cells can be used.

In certain embodiments, a polypeptide or agent described herein is capable of inhibiting tumor growth. In certain embodiments, the polypeptide or agent is capable of inhibiting tumor growth in vivo (e.g., in a mouse model and/or in a human having cancer).

In certain embodiments, a polypeptide or agent described herein is capable of reducing the tumorigenicity of a tumor. In certain embodiments, the polypeptide or agent is capable of reducing the tumorigenicity of a tumor in an animal model, such as a mouse model. In certain embodiments, the polypeptide or agent is capable of reducing the tumorigenicity of a tumor comprising cancer stem cells in an animal model, such as a mouse model. In certain embodiments, the number or frequency of cancer stem cells in a tumor is reduced by at least about two-fold, about three-fold, about five-fold, about ten-fold, about 50-fold, about 100-fold, or about 1000-fold. In certain embodiments, the reduction in the number or frequency of cancer stem cells is determined by limiting dilution assay using an animal model. Additional examples and guidance regarding the use of limiting dilution assays to determine a reduction in the number or frequency of cancer stem cells in a tumor can be found, e.g., in International Publication Number WO 2008/042236; U.S. Patent Publication No. 2008/0064049; and U.S. Patent Publication No. 2008/0178305.

In certain embodiments, a polypeptide or agent described herein has one or more of the following effects: inhibits proliferation of tumor cells, inhibits tumor growth, reduces the tumorigenicity of a tumor, reduces the tumorigenicity of a tumor by reducing the frequency of cancer stem cells in the tumor, triggers cell death of tumor cells, increases cell contact-dependent growth inhibition, increases tumor cell apoptosis, reduces epithelial mesenchymal transition (EMT), or decreases survival of tumor cells. In some embodiments, the polypeptide or agent has one or more of the following effects: inhibits viral infection, inhibits chronic viral infection, reduces viral load, triggers cell death of virus-infected cells, or reduces the number or percentage of virus-infected cells.

In certain embodiments, a polypeptide or agent described herein has a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 2 days, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks. In certain embodiments, the polypeptide or agent is an IgG (e.g., IgG1 or IgG2) fusion protein that has a circulating half-life in mice, rats, cynomolgus monkeys, or humans of at least about 5 hours, at least about 10 hours, at least about 24 hours, at least about 3 days, at least about 1 week, at least about 2 weeks, or at least 3 weeks. Methods of increasing (or decreasing) the half-life of agents such as polypeptides and soluble receptors are known in the art. For example, known methods of increasing the circulating half-life of IgG fusion proteins include the introduction of mutations in the Fc region which increase the pH-dependent binding of the antibody to the neonatal Fc receptor (FcRn) at pH 6.0. Known methods of increasing the circulating half-life of soluble receptors lacking a Fc region include such techniques as PEGylation.

In some embodiments of the present invention, the agent is a polypeptide. The polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide that binds GITR. The polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide that binds OX40. The polypeptide can be a recombinant polypeptide, a natural polypeptide, or a synthetic polypeptide that binds CD40. It will be recognized in the art that some amino acid sequences of the invention can be varied without significant effect of the structure or function of the protein. Thus, the invention further includes variations of the polypeptides which show substantial binding activity to GITR. The invention also includes variations of the polypeptides which show substantial binding activity to OX40. The invention also includes variations of the polypeptides which show substantial binding activity to CD40. In some embodiments, amino acid sequence variations of the polypeptides include deletions, insertions, inversions, repeats, and/or other types of substitutions.

The polypeptides, analogs and variants thereof, can be further modified to contain additional chemical moieties not normally part of the polypeptide. The derivatized moieties can improve the solubility, the biological half-life, and/or absorption of the polypeptide. The moieties can also reduce or eliminate undesirable side effects of the polypeptides and variants. An overview for chemical moieties can be found in Remington: The Science and Practice of Pharmacy, 22^(nd) Edition, 2012, Pharmaceutical Press, London.

The polypeptides described herein can be produced by any suitable method known in the art. Such methods range from direct protein synthesis methods to constructing a DNA sequence encoding polypeptide sequences and expressing those sequences in a suitable host. In some embodiments, a DNA sequence is constructed using recombinant technology by isolating or synthesizing a DNA sequence encoding a wild-type protein of interest. Optionally, the sequence can be mutagenized by site-specific mutagenesis to provide functional analogs thereof.

In some embodiments, a DNA sequence encoding a polypeptide of interest may be constructed by chemical synthesis using an oligonucleotide synthesizer. Oligonucleotides can be designed based on the amino acid sequence of the desired polypeptide and selecting those codons that are favored in the host cell in which the recombinant polypeptide of interest will be produced. Standard methods can be applied to synthesize a polynucleotide sequence encoding an isolated polypeptide of interest. For example, a complete amino acid sequence can be used to construct a back-translated gene. Further, a DNA oligomer containing a nucleotide sequence coding for the particular isolated polypeptide can be synthesized. For example, several small oligonucleotides coding for portions of the desired polypeptide can be synthesized and then ligated. The individual oligonucleotides typically contain 5′ or 3′ overhangs for complementary assembly.

Once assembled (by synthesis, site-directed mutagenesis, or another method), the polynucleotide sequences encoding a particular polypeptide of interest can be inserted into an expression vector and operatively linked to an expression control sequence appropriate for expression of the protein in a desired host. Proper assembly can be confirmed by nucleotide sequencing, restriction enzyme mapping, and/or expression of a biologically active polypeptide in a suitable host. As is well-known in the art, in order to obtain high expression levels of a transfected gene in a host, the gene must be operatively linked to transcriptional and translational expression control sequences that are functional in the chosen expression host.

In certain embodiments, a recombinant expression vector is used to amplify and express DNA encoding a polypeptide or agent described herein. For example, a recombinant expression vector can be a replicable DNA construct which has synthetic or cDNA-derived DNA fragments encoding a polypeptide chain of an agent operatively linked to suitable transcriptional and/or translational regulatory elements derived from mammalian, microbial, viral or insect genes. A transcriptional unit generally comprises an assembly of (1) a genetic element or elements having a regulatory role in gene expression, for example, transcriptional promoters or enhancers, (2) a structural or coding sequence which is transcribed into mRNA and translated into protein, and (3) appropriate transcription and translation initiation and termination sequences. Regulatory elements can include an operator sequence to control transcription. The ability to replicate in a host, usually conferred by an origin of replication, and a selection gene to facilitate recognition of transformants can additionally be incorporated. DNA regions are “operatively linked” when they are functionally related to each other. For example, DNA for a signal peptide (secretory leader) is operatively linked to DNA for a polypeptide if it is expressed as a precursor which participates in the secretion of the polypeptide; a promoter is operatively linked to a coding sequence if it controls the transcription of the sequence; or a ribosome binding site is operatively linked to a coding sequence if it is positioned so as to permit translation. In some embodiments, structural elements intended for use in yeast expression systems include a leader sequence enabling extracellular secretion of translated protein by a host cell. In other embodiments, where recombinant protein is expressed without a leader or transport sequence, it can include an N-terminal methionine residue. This residue can optionally be subsequently cleaved from the expressed recombinant protein to provide a final product.

The choice of an expression control sequence and an expression vector depends upon the choice of host. A wide variety of expression host/vector combinations can be employed. Useful expression vectors for eukaryotic hosts include, for example, vectors comprising expression control sequences from SV40, bovine papilloma virus, adenovirus, and cytomegalovirus. Useful expression vectors for bacterial hosts include known bacterial plasmids, such as plasmids from E. coli, including pCR1, pBR322, pMB9 and their derivatives, and wider host range plasmids, such as M13 and other filamentous single-stranded DNA phages.

Suitable host cells for expression of a polypeptide (or a protein to use as a target) include prokaryotes, yeast cells, insect cells, or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, for example E. coli or Bacillus. Higher eukaryotic cells include established cell lines of mammalian origin as described below. Cell-free translation systems may also be employed. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are well known by those skilled in the art.

Various mammalian cell culture systems are used to express recombinant polypeptides. Expression of recombinant proteins in mammalian cells can be preferred because such proteins are generally correctly folded, appropriately modified, and biologically functional. Examples of suitable mammalian host cell lines include COS-7 (monkey kidney-derived), L-929 (murine fibroblast-derived), C127 (murine mammary tumor-derived), 3T3 (murine fibroblast-derived), CHO (Chinese hamster ovary-derived), HeLa (human cervical cancer-derived), BHK (hamster kidney fibroblast-derived), and HEK-293 (human embryonic kidney-derived) cell lines and variants thereof. Mammalian expression vectors can comprise non-transcribed elements such as an origin of replication, a suitable promoter and enhancer linked to the gene to be expressed, and other 5′ or 3′ flanking non-transcribed sequences, and 5′ or 3′ non-translated sequences, such as necessary ribosome binding sites, a polyadenylation site, splice donor and acceptor sites, and transcriptional termination sequences.

Expression of recombinant proteins in insect cell culture systems (e.g., baculovirus) also offers a robust method for producing correctly folded and biologically functional proteins. Baculovirus systems for production of heterologous proteins in insect cells are well-known to those of skill in the art.

Thus, the present invention provides cells comprising the polypeptides and agents described herein. In some embodiments, the cells produce the polypeptides and agents described herein. In certain embodiments, the cells produce a fusion protein. In some embodiments, the cells produce a soluble receptor/ligand. In some embodiments, the cells produce an antibody. In some embodiments, the cells produce a bispecific agent. In some embodiments, the cells produce a bispecific antibody. In some embodiments, the cells produce a homodimeric bispecific agent. In some embodiments, the cells produce a heterodimeric bispecific agent.

The proteins produced by a transformed host can be purified according to any suitable method. Standard methods include chromatography (e.g., ion exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for protein purification. Affinity tags such as hexa-histidine, maltose binding domain, influenza coat sequence, and glutathione-S-transferase can be attached to the protein to allow easy purification by passage over an appropriate affinity column. Isolated proteins can also be physically characterized using such techniques as proteolysis, mass spectrometry (MS), nuclear magnetic resonance (NMR), high performance liquid chromatography (HPLC), and x-ray crystallography.

In some embodiments, supernatants from expression systems which secrete recombinant protein into culture media can be first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a suitable purification matrix. In some embodiments, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose, or other types commonly employed in protein purification. In some embodiments, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. In some embodiments, a hydroxyapatite media can be employed, including but not limited to, ceramic hydroxyapatite (CHT). In certain embodiments, one or more reverse-phase HPLC steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify a polypeptide or agent. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a homogeneous recombinant protein.

In some embodiments, recombinant protein produced in bacterial culture can be isolated, for example, by initial extraction from cell pellets, followed by one or more concentration, salting-out, aqueous ion exchange, or size exclusion chromatography steps. HPLC can be employed for final purification steps. Microbial cells employed in expression of a recombinant protein can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

In certain embodiments, a polypeptide or agent described herein is a polypeptide that does not comprise an immunoglobulin Fc region. In certain embodiments, the polypeptide comprises a protein scaffold of a type selected from the group consisting of protein A, protein G, a lipocalin, a fibronectin domain, an ankyrin consensus repeat domain, and thioredoxin. A variety of methods for identifying and producing non-antibody polypeptides that bind with high affinity to a protein target are known in the art. In certain embodiments, phage display technology may be used to produce and/or identify a binding polypeptide. In certain embodiments, mammalian cell display technology may be used to produce and/or identify a binding polypeptide.

It can further be desirable to modify a polypeptide in order to increase (or decrease) its serum half-life. This can be achieved, for example, by incorporation of a salvage receptor binding epitope into the polypeptide by mutation of the appropriate region in the polypeptide or by incorporating the epitope into a peptide tag that is then fused to the polypeptide at either end or in the middle (e.g., by DNA or peptide synthesis).

Heteroconjugate molecules are also within the scope of the present invention. Heteroconjugate molecules are composed of two covalently joined polypeptides. Such molecules have, for example, been proposed to target immune cells to unwanted cells, such as tumor cells. It is also contemplated that the heteroconjugate molecules can be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

In certain embodiments, a polypeptide or agent described herein can be used in any one of a number of conjugated (i.e. an immunoconjugate or radioconjugate) or non-conjugated forms. In certain embodiments, the polypeptides or agents can be used in a non-conjugated form to harness the subject's natural defense mechanisms including CDC and ADCC to eliminate malignant or cancer cells.

In certain embodiments, an agent described herein is a small molecule. The term “small molecule” generally refers to a low molecular weight organic compound which is by definition not a peptide/protein.

In some embodiments, a polypeptide or agent described herein is conjugated to a cytotoxic agent. In some embodiments, the cytotoxic agent is a chemotherapeutic agent including, but not limited to, methotrexate, adriamicin, doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents. In some embodiments, the cytotoxic agent is an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof, including, but not limited to, diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain, ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. In some embodiments, the cytotoxic agent is a radioisotope to produce a radioconjugate or a radioconjugated agent. A variety of radionuclides are available for the production of radioconjugated agents including, but not limited to ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I, ¹¹¹In, ¹³¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, and ²¹²Bi. Conjugates of a polypeptide or agent and one or more small molecule toxins, such as a calicheamicin, maytansinoids, a trichothene, and CC 1065, and the derivatives of these toxins that have toxin activity, can also be used. Conjugates of a polypeptide or agent and cytotoxic agent are made using a variety of bifunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyidithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene).

III. POLYNUCLEOTIDES

In certain embodiments, the invention encompasses polynucleotides comprising polynucleotides that encode a polypeptide or agent described herein. The term “polynucleotides that encode a polypeptide” encompasses a polynucleotide which includes only coding sequences for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequences. The polynucleotides of the invention can be in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA, and synthetic DNA; and can be double-stranded or single-stranded, and if single stranded can be the coding strand or non-coding (anti-sense) strand.

In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81. In certain embodiments, the polynucleotide comprises a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106. In some embodiments, the polynucleotide comprises a polynucleotide comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:73, and SEQ ID NO:86. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the GITR-binding agents described herein. In some embodiments, the polynucleotide is a plasmid “hGITRL-hIgG1” deposited with ATCC at 10801 University Boulevard, Manassas, Va., USA, under the conditions of the Budapest Treaty on Apr. 21, 2015 and assigned designation number PTA-122112. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the OX40-binding agents described herein. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the CD40-binding agents described herein. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the GITR-binding agents described herein and a signal sequence. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the OX40-binding agents described herein and a signal sequence. In some embodiments, a polynucleotide comprises a polynucleotide that encodes a polypeptide of any of the CD40-binding agents described herein and a signal sequence. In some embodiments, a vector comprises the polynucleotide. In some embodiments, a cell comprises the polynucleotide. In some embodiments, a cell comprises the vector. In some embodiments, the cell is isolated.

In certain embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. In certain embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:43, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81. In certain embodiments, a polynucleotide comprises a polynucleotide having a nucleotide sequence at least 80% identical, at least 85% identical, at least 90% identical, at least 95% identical, and in some embodiments, at least 96%, 97%, 98% or 99% identical to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106.

Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9. Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, and SEQ ID NO:81. Also provided is a polynucleotide that comprises a polynucleotide that hybridizes to a polynucleotide encoding an amino acid sequence selected from the group consisting of: SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, and SEQ ID NO:106. In certain embodiments, the hybridization is under conditions of high stringency. Conditions of high stringency are known to those of skill in the art and may include but are not limited to, (1) employ low ionic strength and high temperature for washing, for example 15 mM sodium chloride/1.5 mM sodium citrate (lx SSC) with 0.1% sodium dodecyl sulfate at 50° C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1% Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 in 5×SSC (0.75M NaCl, 75 mM sodium citrate) at 42° C.; or (3) employ 50% formamide, 5×SSC, 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42° C., with washes in 0.2×SSC containing 50% formamide at 55° C., followed by a high-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.

In certain embodiments, a polynucleotide comprises the coding sequence for the mature polypeptide fused in the same reading frame to a polynucleotide which aids, for example, in expression and secretion of a polypeptide from a host cell (e.g., a leader sequence which functions as a secretory sequence for controlling transport of a polypeptide from the cell). The polypeptide having a leader sequence is a pre-protein and can have the leader sequence cleaved by the host cell to form the mature form of the polypeptide. The polynucleotides can also encode for a pro-protein which is the mature protein plus additional 5′ amino acid residues. A mature protein having a pro-sequence is a pro-protein and is an inactive form of the protein. Once the pro-sequence is cleaved an active mature protein remains.

In certain embodiments, a polynucleotide comprises the coding sequence for the mature polypeptide fused in the same reading frame to a marker sequence that allows, for example, for purification of the encoded polypeptide. For example, the marker sequence can be a hexa-histidine tag supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or the marker sequence can be a hemagglutinin (HA) tag derived from the influenza hemagglutinin protein when a mammalian host (e.g., COS-7 cells) is used. In some embodiments, the marker sequence is a FLAG-tag, a peptide of sequence DYKDDDDK (SEQ ID NO:39) which can be used in conjunction with other affinity tags.

The present invention further relates to variants of the hereinabove described polynucleotides encoding, for example, fragments, analogs, and/or derivatives.

In certain embodiments, the present invention provides a polynucleotide comprising a polynucleotide having a nucleotide sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, and in some embodiments, at least about 96%, 97%, 98% or 99% identical to a polynucleotide encoding a polypeptide comprising a polypeptide or agent described herein.

As used herein, the phrase a polynucleotide having a nucleotide sequence at least, for example, 95% “identical” to a reference nucleotide sequence is intended to mean that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence can include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence can be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5′ or 3′ terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.

The polynucleotide variants can contain alterations in the coding regions, non-coding regions, or both. In some embodiments, a polynucleotide variant contains alterations which produce silent substitutions, additions, or deletions, but does not alter the properties or activities of the encoded polypeptide. In some embodiments, a polynucleotide variant comprises silent substitutions that results in no change to the amino acid sequence of the polypeptide (due to the degeneracy of the genetic code). Polynucleotide variants can be produced for a variety of reasons, for example, to optimize codon expression for a particular host (i.e., change codons in the human mRNA to those preferred by a bacterial host such as E. coli). In some embodiments, a polynucleotide variant comprises at least one silent mutation in a non-coding or a coding region of the sequence.

In some embodiments, a polynucleotide variant is produced to modulate or alter expression (or expression levels) of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to increase expression of the encoded polypeptide. In some embodiments, a polynucleotide variant is produced to decrease expression of the encoded polypeptide. In some embodiments, a polynucleotide variant has increased expression of the encoded polypeptide as compared to a parental polynucleotide sequence. In some embodiments, a polynucleotide variant has decreased expression of the encoded polypeptide as compared to a parental polynucleotide sequence.

In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a heterodimeric molecule. In some embodiments, at least one polynucleotide variant is produced (without changing the amino acid sequence of the encoded polypeptide) to increase production of a bispecific agent, a bispecific antibody, or a heterodimeric agent.

In certain embodiments, the polynucleotides are isolated. In certain embodiments, the polynucleotides are substantially pure.

Vectors and cells comprising the polynucleotides described herein are also provided. In some embodiments, an expression vector comprises a polynucleotide molecule. In some embodiments, a host cell comprises an expression vector comprising the polynucleotide molecule. In some embodiments, a host cell comprises a polynucleotide molecule.

IV. METHODS OF USE AND PHARMACEUTICAL COMPOSITIONS

The polypeptides or agents of the invention are useful in a variety of applications including, but not limited to, therapeutic treatment methods, such as immunotherapy for cancer. In certain embodiments, a polypeptide or agent described herein is useful for activating, promoting, increasing, and/or enhancing an immune response, inhibiting tumor growth, reducing tumor volume, inducing tumor regression, increasing tumor cell apoptosis, and/or reducing the tumorigenicity of a tumor. In certain embodiments, the polypeptides or agents of the invention are also useful for immunotherapy against pathogens, such as viruses. In certain embodiments, a polypeptide or agent described herein is useful for inhibiting viral infection, reducing viral infection, increasing virally-infected cell apoptosis, and/or increasing killing of virus-infected cells. The methods of use may be in vitro, ex vivo, or in vivo methods.

The present invention provides methods for activating an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for promoting an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for increasing an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the invention provides methods for enhancing an immune response in a subject using a polypeptide or agent described herein. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing cell-mediated immunity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing Th1-type responses. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD4+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CD8+ T-cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CTL activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing T-cell activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing CU activity and increasing NK cell activity. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of Treg cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises inhibiting or decreasing the suppressive activity of MDSCs. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing the number of the percentage of memory T-cells. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term immune memory function. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises increasing long-term memory. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of substantial side effects and/or immune-based toxicities. In some embodiments, the activating, promoting, increasing, and/or enhancing of an immune response comprises no evidence of cytokine release syndrome (CRS) or a cytokine storm. In some embodiments, the immune response is a result of antigenic stimulation. In some embodiments, the antigenic stimulation is a tumor cell. In some embodiments, the antigenic stimulation is cancer. In some embodiments, the antigenic stimulation is a pathogen. In some embodiments, the antigenic stimulation is a virally-infected cell.

In vivo and in vitro assays for determining whether an agent or polypeptide modulates, activates, or inhibits an immune response are known in the art or are being developed.

In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent binds human GITR. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to GITR. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain GITRL trimer.

In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent binds human OX40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to OX40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain OX40L trimer.

In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent binds human CD40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of increasing an immune response in a subject comprises administering to the subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain CD40L trimer.

In certain embodiments of the methods described herein, a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human GITR. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to GITR. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain GITRL trimer. In some embodiments, the polypeptide comprises a single chain GITRL trimer and a Fc region. In some embodiments, the polypeptide is 336B3. In some embodiments, the polypeptide is 336B11.

In certain embodiments of the methods described herein, a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human OX40. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to OX40. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain OX40L trimer. In some embodiments, the polypeptide comprises a single chain OX40L trimer and a Fc region.

In certain embodiments of the methods described herein, a method of activating or enhancing a persistent or long-term immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of activating or enhancing a persistent immune response to a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain CD40L trimer. In some embodiments, the polypeptide comprises a single chain CD40L trimer and a Fc region.

In certain embodiments of the methods described herein, a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human GITR. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to GITR. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain GITRL trimer. In some embodiments, the polypeptide comprises a single chain GITRL trimer and a Fc region. In some embodiments, the polypeptide is 336B3. In some embodiments, the polypeptide is 336B11.

In certain embodiments of the methods described herein, a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human OX40. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to OX40. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain OX40L trimer. In some embodiments, the polypeptide comprises a single chain OX40L trimer and a Fc region.

In certain embodiments of the methods described herein, a method of inducing a persistent or long-term immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of inducing a persistent immunity which inhibits tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain CD40L trimer. In some embodiments, the polypeptide comprises a single chain CD40L trimer and a Fc region.

In certain embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human GITR. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to GITR. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain GITRL trimer. In some embodiments, the polypeptide comprises a single chain GITRL trimer and a Fc region. In some embodiments, the polypeptide is 336B3. In some embodiments, the polypeptide is 336B11.

In certain embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human OX40. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to OX40. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain OX40L trimer. In some embodiments, the polypeptide comprises a single chain OX40L trimer and a Fc region.

In certain embodiments of the methods described herein, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent which binds human CD40. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent is a single chain fusion polypeptide that specifically binds to CD40. In some embodiments, a method of inhibiting tumor relapse or tumor regrowth comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein, wherein the polypeptide or agent comprises a single chain CD40L trimer. In some embodiments, the polypeptide comprises a single chain CD40L trimer and a Fc region.

The present invention also provides methods for inhibiting growth of a tumor using a polypeptide or agent described herein. In certain embodiments, the method of inhibiting growth of a tumor comprises contacting a cell mixture with a polypeptide or agent in vitro. For example, an immortalized cell line or a cancer cell line mixed with immune cells (e.g., T-cells, cytolytic T-cells, or NK cells) is cultured in medium to which is added a test agent. In some embodiments, tumor cells are isolated from a patient sample such as, for example, a tissue biopsy, pleural effusion, or blood sample, mixed with immune cells (e.g., T-cells, cytolytic T-cell, and/or NK cells), and cultured in medium to which is added a test agent. In some embodiments, the polypeptide or agent increases, promotes, and/or enhances the activity of the immune cells. In some embodiments, the polypeptide or agent inhibits tumor cell growth.

In some embodiments, the method of inhibiting growth of a tumor comprises contacting the tumor or tumor cells with a polypeptide or agent described herein in vivo. In certain embodiments, contacting a tumor or tumor cell with a polypeptide or agent is undertaken in an animal model. For example, a test agent may be administered to mice which have tumors. In some embodiments, the polypeptide or agent increases, promotes, and/or enhances the activity of immune cells in the mice. In some embodiments, the polypeptide or agent inhibits tumor growth. In some embodiments, the polypeptide or agent is administered at the same time or shortly after introduction of tumor cells into the animal to prevent tumor growth (“preventative model”). In some embodiments, the polypeptide or agent is administered as a therapeutic after tumors have grown to a specified size (“therapeutic model”).

In certain embodiments, the method of inhibiting growth of a tumor comprises administering to a subject a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a tumor or the subject had a tumor which was removed.

In addition, the invention provides a method of inhibiting growth of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the tumor comprises cancer stem cells. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of the polypeptide or agent. In some embodiments, a method of reducing the frequency of cancer stem cells in a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent is provided. In some embodiments, the polypeptide is 336B3. In some embodiments, the polypeptide is 336B11.

In addition, the invention provides a method of reducing the tumorigenicity of a tumor in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the tumor comprises cancer stem cells. In some embodiments, the tumorigenicity of a tumor is reduced by reducing the frequency of cancer stem cells in the tumor. In some embodiments, the methods comprise using the polypeptides or agents described herein. In certain embodiments, the frequency of cancer stem cells in the tumor is reduced by administration of a polypeptide or agent.

In some embodiments, the tumor is a solid tumor. In certain embodiments, the tumor is a tumor selected from the group consisting of: colorectal tumor, pancreatic tumor, lung tumor, ovarian tumor, liver tumor, breast tumor, kidney tumor, prostate tumor, neuroendocrine tumor, gastrointestinal tumor, melanoma, cervical tumor, bladder tumor, glioblastoma, and head and neck tumor. In certain embodiments, the tumor is a colorectal tumor. In certain embodiments, the tumor is an ovarian tumor. In some embodiments, the tumor is a lung tumor. In certain embodiments, the tumor is a pancreatic tumor. In certain embodiments, the tumor is a melanoma tumor. In some embodiments, the tumor is a bladder tumor.

In some embodiments, the tumor expresses or overexpresses a tumor antigen targeted by the polypeptide or agent, such as a bispecific agent which comprises an antigen-binding site that specifically binds the tumor antigen.

The present invention further provides methods for treating cancer in a subject comprising administering to the subject a therapeutically effective amount of a polypeptide or agent described herein. In some embodiments, the polypeptide or agent binds GITR and inhibits or reduces growth of the cancer. In some embodiments, the polypeptide or agent binds OX40 and inhibits or reduces growth of the cancer. In some embodiments, the polypeptide or agent binds CD40 and inhibits or reduces growth of the cancer. In some embodiments, the polypeptide is 336B3. In some embodiments, the polypeptide is 336B11.

The present invention provides for methods of treating cancer comprising administering to a subject (e.g., a subject in need of treatment) a therapeutically effective amount of a polypeptide or agent described herein. In certain embodiments, the subject is a human. In certain embodiments, the subject has a cancerous tumor. In certain embodiments, the subject has had a tumor removed.

In certain embodiments, the cancer is a cancer selected from the group consisting of colorectal cancer, pancreatic cancer, lung cancer, ovarian cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, neuroendocrine cancer, bladder cancer, brain cancer, glioblastoma, and head and neck cancer. In certain embodiments, the cancer is pancreatic cancer. In certain embodiments, the cancer is ovarian cancer. In certain embodiments, the cancer is colorectal cancer. In certain embodiments, the cancer is breast cancer. In certain embodiments, the cancer is prostate cancer. In certain embodiments, the cancer is lung cancer. In certain embodiments, the cancer is melanoma. In some embodiments, the cancer is bladder cancer.

In some embodiments, the cancer is a hematologic cancer. In some embodiment, the cancer is selected from the group consisting of: acute myelogenous leukemia (AML), Hodgkin lymphoma, multiple myeloma, T-cell acute lymphoblastic leukemia (T-ALL), chronic lymphocytic leukemia (CLL), hairy cell leukemia, chronic myelogenous leukemia (CML), non-Hodgkin lymphoma, diffuse large B-cell lymphoma (DLBCL), mantle cell lymphoma (MCL), and cutaneous T-cell lymphoma (CTCL).

The invention also provides a method of activating or enhancing TNFRSF (e.g., GITR, OX40, or CD40) signaling in a cell comprising contacting the cell with an effective amount of a polypeptide or agent described herein. In some embodiments, a method of activating or enhancing GITR signaling in a cell comprises contacting the cell with an effective amount of a GITR-binding polypeptide or agent described herein. In some embodiments, a method of activating or enhancing OX40 signaling in a cell comprises contacting the cell with an effective amount of an OX40-binding polypeptide or agent described herein. In some embodiments, a method of activating or enhancing CD40 signaling in a cell comprises contacting the cell with an effective amount of a CD40-binding polypeptide or agent described herein. In some embodiments, the polypeptide is 336B3. In some embodiments, the polypeptide is 336B11. In certain embodiments, the cell is a T-cell. In some embodiments, the cell is a cytolytic cell. In some embodiments, the cell is a CTL. In some embodiments, the cell is a NK cell. In certain embodiments, the method is an in vivo method wherein the step of contacting the cell with the polypeptide or agent comprises administering a therapeutically effective amount of the polypeptide or agent to the subject. In some embodiments, the method is an in vitro or ex vivo method.

The present invention provides methods of determining the level of expression of TNFRSF and/or TNFSF. In some embodiments, the level of expression of GITR is determined. In some embodiments, the level of expression of GITRL is determined. In some embodiments, the level of expression of OX40 is determined. In some embodiments, the level of expression of OX40L is determined. In some embodiments, the level of expression of CD40 is determined. In some embodiments, the level of expression of CD40L is determined. Methods for determining the level of nucleic acid expression in a cell, tumor, or cancer are known by those of skill in the art. These methods include, but are not limited to, PCR-based assays, microarray analyses, and nucleotide sequencing (e.g., NextGen sequencing). Methods for determining the level of protein expression in a cell, tumor, or cancer include, but are not limited to, Western blot analyses, protein arrays, ELISAs, immunohistochemistry (IHC), and FACS.

Methods for determining whether a tumor or cancer has an elevated level of expression of a nucleic acid or protein can use a variety of samples. In some embodiments, the sample is taken from a subject having a tumor or cancer. In some embodiments, the sample is a fresh tumor/cancer sample. In some embodiments, the sample is a frozen tumor/cancer sample. In some embodiments, the sample is a formalin-fixed paraffin-embedded sample. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a plasma sample. In some embodiments, the sample is processed to a cell lysate. In some embodiments, the sample is processed to DNA or RNA.

The present invention provides compositions comprising a polypeptide or agent described herein. The present invention also provides pharmaceutical compositions comprising a polypeptide or agent described herein and a pharmaceutically acceptable vehicle. In some embodiments, the pharmaceutical compositions find use in immunotherapy. In some embodiments, the pharmaceutical compositions find use in immuno-oncology. In some embodiments, the compositions find use in inhibiting tumor growth. In some embodiments, the pharmaceutical compositions find use in inhibiting tumor growth in a subject (e.g., a human patient). In some embodiments, the compositions find use in treating cancer. In some embodiments, the pharmaceutical compositions find use in treating cancer in a subject (e.g., a human patient).

Formulations are prepared for storage and use by combining a purified agent of the present invention with a pharmaceutically acceptable vehicle (e.g., a carrier or excipient). Those of skill in the art generally consider pharmaceutically acceptable carriers, excipients, and/or stabilizers to be inactive ingredients of a formulation or pharmaceutical composition.

In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20 mM histidine, 40 mM NaCl, 5% sucrose, and 0.01% polysorbate 20. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20 mM histidine, 40 mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at pH 5.5. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20 mM histidine, 40 mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at pH 6.0. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20 mM histidine, 40 mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at pH 6.5. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 20 mM histidine, 100 mM NaCl, 150 mM sucrose, and 0.01% polysorbate 20 at pH 6.0. In some embodiments, the polypeptides or agents described herein are formulated in a buffer comprising of 10 mM potassium phosphate and 0.04% polysorbate 20 at pH 7.5.

Thus, in some embodiments the invention provides compositions or pharmaceutical compositions comprising a polypeptide or agent described herein and further comprising about 20 mM histidine, about 40 mM NaCl, about 5% sucrose, and about 0.01% polysorbate 20. In some embodiments the pH of the composition is about pH 5.5, about pH 6.0, or about pH 6.5.

In some embodiments, a polypeptide or agent described herein is lyophilized and/or stored in a lyophilized form. In some embodiments, a formulation comprising a polypeptide or agent described herein is lyophilized.

Suitable pharmaceutically acceptable vehicles include, but are not limited to, nontoxic buffers such as phosphate, citrate, and other organic acids; salts such as sodium chloride; antioxidants including ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens, such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight polypeptides (e.g., less than about 10 amino acid residues); proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; carbohydrates such as monosaccharides, disaccharides, glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as TWEEN or polyethylene glycol (PEG). (Remington: The Science and Practice of Pharmacy, 22^(nd) Edition, 2012, Pharmaceutical Press, London.).

The pharmaceutical compositions of the present invention can be administered in any number of ways for either local or systemic treatment. Administration can be topical by epidermal or transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders; pulmonary by inhalation or insufflation of powders or aerosols, including by nebulizer, intratracheal, and intranasal; oral; or parenteral including intravenous, intraarterial, intratumoral, subcutaneous, intraperitoneal, intramuscular (e.g., injection or infusion), or intracranial (e.g., intrathecal or intraventricular).

The therapeutic formulation can be in unit dosage form. Such formulations include tablets, pills, capsules, powders, granules, solutions or suspensions in water or non-aqueous media, or suppositories. In solid compositions such as tablets the principal active ingredient is mixed with a pharmaceutical carrier. Conventional tableting ingredients include corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and diluents (e.g., water). These can be used to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. The solid preformulation composition is then subdivided into unit dosage forms of a type described above. The tablets, pills, etc. of the formulation or composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner composition covered by an outer component. Furthermore, the two components can be separated by an enteric layer that serves to resist disintegration and permits the inner component to pass intact through the stomach or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials include a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol and cellulose acetate.

The polypeptides or agents described herein can also be entrapped in microcapsules. Such microcapsules are prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions as described in Remington: The Science and Practice of Pharmacy, 22^(nd) Edition, 2012, Pharmaceutical Press, London.

In certain embodiments, pharmaceutical formulations include a polypeptide or agent of the present invention complexed with liposomes. Methods to produce liposomes are known to those of skill in the art. For example, some liposomes can be generated by reverse phase evaporation with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes can be extruded through filters of defined pore size to yield liposomes with the desired diameter.

In certain embodiments, sustained-release preparations comprising the polypeptides or agents described herein can be produced. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing a polypeptide or agent, where the matrices are in the form of shaped articles (e.g., films or microcapsules). Examples of sustained-release matrices include polyesters, hydrogels such as poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol), polylactides, copolymers of L-glutamic acid and 7 ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

In certain embodiments, in addition to administering a polypeptide or agent described herein, the method or treatment further comprises administering at least one additional immune response stimulating agent. In some embodiments, the additional immune response stimulating agent includes, but is not limited to, a colony stimulating factor (e.g., granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), stem cell factor (SCF)), an interleukin (e.g., IL-1, IL2, IL-3, IL-7, IL-12, IL-15, IL-18), a checkpoint inhibitor, an antibody that blocks immunosuppressive functions (e.g., an anti-CTLA-4 antibody, anti-CD28 antibody, anti-CD3 antibody), a toll-like receptor (e.g., TLR4, TLR7, TLR9), or a member of the B7 family (e.g., CD80, CD86). An additional immune response stimulating agent can be administered prior to, concurrently with, and/or subsequently to, administration of the polypeptide or agent. Pharmaceutical compositions comprising a polypeptide or agent and the immune response stimulating agent(s) are also provided. In some embodiments, the immune response stimulating agent comprises 1, 2, 3, or more immune response stimulating agents.

In certain embodiments, in addition to administering a polypeptide or agent described herein, the method or treatment further comprises administering at least one additional therapeutic agent. An additional therapeutic agent can be administered prior to, concurrently with, and/or subsequently to, administration of the polypeptide or agent. Pharmaceutical compositions comprising a polypeptide or agent and the additional therapeutic agent(s) are also provided. In some embodiments, the at least one additional therapeutic agent comprises 1, 2, 3, or more additional therapeutic agents.

Combination therapy with two or more therapeutic agents often uses agents that work by different mechanisms of action, although this is not required. Combination therapy using agents with different mechanisms of action may result in additive or synergetic effects. Combination therapy may allow for a lower dose of each agent than is used in monotherapy, thereby reducing toxic side effects and/or increasing the therapeutic index of the polypeptide or agent(s). Combination therapy may decrease the likelihood that resistant cancer cells will develop. In some embodiments, combination therapy comprises a therapeutic agent that affects the immune response (e.g., enhances or activates the response) and a therapeutic agent that affects (e.g., inhibits or kills) the tumor/cancer cells.

In some embodiments of the methods described herein, the combination of a polypeptide or agent described herein and at least one additional therapeutic agent results in additive or synergistic results. In some embodiments, the combination therapy results in an increase in the therapeutic index of the polypeptide or agent. In some embodiments, the combination therapy results in an increase in the therapeutic index of the additional therapeutic agent(s). In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the polypeptide or agent. In some embodiments, the combination therapy results in a decrease in the toxicity and/or side effects of the additional therapeutic agent(s).

Useful classes of therapeutic agents include, for example, anti-tubulin agents, auristatins, DNA minor groove binders, DNA replication inhibitors, alkylating agents (e.g., platinum complexes such as cisplatin, mono(platinum), bis(platinum) and tri-nuclear platinum complexes and carboplatin), anthracyclines, antibiotics, anti-folates, anti-metabolites, chemotherapy sensitizers, duocarmycins, etoposides, fluorinated pyrimidines, ionophores, lexitropsins, nitrosoureas, platinols, purine antimetabolites, puromycins, radiation sensitizers, steroids, taxanes, topoisomerase inhibitors, vinca alkaloids, or the like. In certain embodiments, the second therapeutic agent is an alkylating agent, an antimetabolite, an antimitotic, a topoisomerase inhibitor, or an angiogenesis inhibitor.

Therapeutic agents that may be administered in combination with the polypeptides or agents described herein include chemotherapeutic agents. Thus, in some embodiments, the method or treatment involves the administration of a polypeptide or agent of the present invention in combination with a chemotherapeutic agent or in combination with a cocktail of chemotherapeutic agents. Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of chemotherapies. Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously. Preparation and dosing schedules for such chemotherapeutic agents can be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in The Chemotherapy Source Book, 4^(th) Edition, 2008, M. C. Perry, Editor, Lippincott, Williams & Wilkins, Philadelphia, Pa.

Chemotherapeutic agents useful in the present invention include, but are not limited to, alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaoramide and trimethylolomelamime; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, calicheamicin, carabicin, caminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytosine arabinoside, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenishers such as folinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elformithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK; razoxane; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2,2′,2″-trichlorotriethylamine; urethan; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside (Ara-C); taxoids, e.g. paclitaxel (TAXOL) and docetaxel (TAXOTERE); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; ibandronate; CPT11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoic acid; esperamicins; capecitabine (XELODA); and pharmaceutically acceptable salts, acids or derivatives of any of the above. Chemotherapeutic agents also include anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (FARESTON); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above. In certain embodiments, the additional therapeutic agent is cisplatin. In certain embodiments, the additional therapeutic agent is carboplatin.

In certain embodiments of the methods described herein, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerase inhibitors are chemotherapy agents that interfere with the action of a topoisomerase enzyme (e.g., topoisomerase I or II). Topoisomerase inhibitors include, but are not limited to, doxorubicin HCl, daunorubicin citrate, mitoxantrone HCl, actinomycin D, etoposide, topotecan HCl, teniposide (VM-26), and irinotecan, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In some embodiments, the additional therapeutic agent is irinotecan.

In certain embodiments, the chemotherapeutic agent is an anti-metabolite. An anti-metabolite is a chemical with a structure that is similar to a metabolite required for normal biochemical reactions, yet different enough to interfere with one or more normal functions of cells, such as cell division. Anti-metabolites include, but are not limited to, gemcitabine, fluorouracil, capecitabine, methotrexate sodium, ralitrexed, pemetrexed, tegafur, cytosine arabinoside, thioguanine, 5-azacytidine, 6-mercaptopurine, azathioprine, 6-thioguanine, pentostatin, fludarabine phosphate, and cladribine, as well as pharmaceutically acceptable salts, acids, or derivatives of any of these. In certain embodiments, the additional therapeutic agent is gemcitabine.

In certain embodiments of the methods described herein, the chemotherapeutic agent is an antimitotic agent, including, but not limited to, agents that bind tubulin. In some embodiments, the agent is a taxane. In certain embodiments, the agent is paclitaxel or docetaxel, or a pharmaceutically acceptable salt, acid, or derivative of paclitaxel or docetaxel. In certain embodiments, the agent is paclitaxel (TAXOL), docetaxel (TAXOTERE), albumin-bound paclitaxel (nab-paclitaxel; ABRAXANE), DHA-paclitaxel, or PG-paclitaxel. In certain alternative embodiments, the antimitotic agent comprises a vinca alkaloid, such as vincristine, vinblastine, vinorelbine, or vindesine, or pharmaceutically acceptable salts, acids, or derivatives thereof. In some embodiments, the antimitotic agent is an inhibitor of kinesin Eg5 or an inhibitor of a mitotic kinase such as Aurora A or Plk1. In certain embodiments, the additional therapeutic agent is paclitaxel. In certain embodiments, the additional therapeutic agent is nab-paclitaxel.

In some embodiments of the methods described herein, an additional therapeutic agent comprises an agent such as a small molecule. For example, treatment can involve the combined administration of a polypeptide or agent of the present invention with a small molecule that acts as an inhibitor against tumor-associated antigens including, but not limited to, EGFR, HER2 (ErbB2), and/or VEGF. In some embodiments, a polypeptide or agent of the present invention is administered in combination with a protein kinase inhibitor selected from the group consisting of: gefitinib (IRESSA), erlotinib (TARCEVA), sunitinib (SUTENT), lapatanib, vandetanib (ZACTIMA), AEE788, CI-1033, cediranib (RECENTIN), sorafenib (NEXAVAR), and pazopanib (GW786034B). In some embodiments, an additional therapeutic agent comprises an mTOR inhibitor.

In certain embodiments of the methods described herein, the additional therapeutic agent is a small molecule that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Hippo pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the mTOR/AKR pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the RSPO/LGR pathway.

In some embodiments of the methods described herein, an additional therapeutic agent comprises a biological molecule, such as an antibody. For example, treatment can involve the combined administration of a polypeptide or agent of the present invention with antibodies against tumor-associated antigens including, but not limited to, antibodies that bind EGFR, HER2/ErbB2, and/or VEGF. In certain embodiments, the additional therapeutic agent is an antibody specific for a cancer stem cell marker. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Notch pathway. In some embodiments, the additional therapeutic agent is an antibody that binds a component of the Wnt pathway. In certain embodiments, the additional therapeutic agent is an antibody that inhibits a cancer stem cell pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Notch pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the Wnt pathway. In some embodiments, the additional therapeutic agent is an inhibitor of the BMP pathway. In some embodiments, the additional therapeutic agent is an antibody that inhibits β-catenin signaling. In certain embodiments, the additional therapeutic agent is an antibody that is an angiogenesis inhibitor (e.g., an anti-VEGF or VEGF receptor antibody). In certain embodiments, the additional therapeutic agent is bevacizumab (AVASTIN), ramucirumab, trastuzumab (HERCEPTIN), pertuzumab (OMNITARG), panitumumab (VECTIBIX), nimotuzumab, zalutumumab, or cetuximab (ERBITUX).

In some embodiments of the methods described herein, the additional therapeutic agent is an antibody that modulates the immune response. In some embodiments, the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or an anti-TIGIT antibody.

Furthermore, treatment with a polypeptide or agent described herein can include combination treatment with other biologic molecules, such as one or more cytokines (e.g., lymphokines, interleukins, tumor necrosis factors, and/or growth factors) or can be accompanied by surgical removal of tumors, removal of cancer cells, or any other therapy deemed necessary by a treating physician. In some embodiments, the additional therapeutic agent is an immune response stimulating agent.

In some embodiments of the methods described herein, the polypeptide or agent can be combined with a growth factor selected from the group consisting of: adrenomedullin (AM), angiopoietin (Ang), BMPs, BDNF, EGF, erythropoietin (EPO), FGF, GDNF, G-CSF, GM-CSF, GDF9, HGF, HDGF, IGF, migration-stimulating factor, myostatin (GDF-8), NGF, neurotrophins, PDGF, thrombopoietin, TGF-α, TGF-β, TNF-α, VEGF, P1GF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, and IL-18.

In some embodiments of the methods described herein, the additional therapeutic agent is an immune response stimulating agent. In some embodiments, the immune response stimulating agent is selected from the group consisting of granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), granulocyte colony stimulating factor (G-CSF), interleukin 3 (IL-3), interleukin 12 (IL-12), interleukin 1 (IL-1), interleukin 2 (IL-2), B7-1 (CD80), B7-2 (CD86), 4-1BB ligand, anti-CD3 antibody, anti-CTLA-4 antibody, anti-TIGIT antibody, anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, and anti-TIM-3 antibody.

In some embodiments of the methods described herein, an immune response stimulating agent is selected from the group consisting of: a modulator of PD-1 activity, a modulator of PD-L1 activity, a modulator of PD-L2 activity, a modulator of CTLA-4 activity, a modulator of CD28 activity, a modulator of CD80 activity, a modulator of CD86 activity, a modulator of 4-1BB activity, an modulator of OX40 activity, a modulator of KIR activity, a modulator of Tim-3 activity, a modulator of LAG3 activity, a modulator of CD27 activity, a modulator of CD40 activity, a modulator of GITR activity, a modulator of TIGIT activity, a modulator of CD20 activity, a modulator of CD96 activity, a modulator of IDO1 activity, a cytokine, a chemokine, an interferon, an interleukin, a lymphokine, a member of the tumor necrosis factor (TNF) family, and an immunostimulatory oligonucleotide.

In some embodiments of the methods described herein, an immune response stimulating agent is selected from the group consisting of: a PD-1 antagonist, a PD-L1 antagonist, a PD-L2 antagonist, a CTLA-4 antagonist, a CD80 antagonist, a CD86 antagonist, a KIR antagonist, a Tim-3 antagonist, a LAG3 antagonist, a TIGIT antagonist, a CD20 antagonist, a CD96 antagonist, and/or an IDO1 antagonist.

In some embodiments of the methods described herein, the PD-1 antagonist is an antibody that specifically binds PD-1. In some embodiments, the antibody that binds PD-1 is KEYTRUDA (MK-3475), pidilizumab (CT-011), nivolumab (OPDIVO, BMS-936558, MDX-1106), MEDI0680 (AMP-514), REGN2810, BGB-A317, PDR-001, or STI-A1110. In some embodiments, the antibody that binds PD-1 is described in PCT Publication WO 2014/179664, for example, an antibody identified as APE2058, APE1922, APE1923, APE1924, APE 1950, or APE1963, or an antibody containing the CDR regions of any of these antibodies. In other embodiments, the PD-1 antagonist is a fusion protein that includes PD-L2, for example, AMP-224. In other embodiments, the PD-1 antagonist is a peptide inhibitor, for example, AUNP-12.

In some embodiments, the PD-L1 antagonist is an antibody that specifically binds PD-L1. In some embodiments, the antibody that binds PD-L1 is atezolizumab (RG7446, MPDL3280A), MEDI4736, BMS-936559 (MDX-1105), avelumab (MSB0010718C), KD033, the antibody portion of KD033, or STI-A1014. In some embodiments, the antibody that binds PD-L1 is described in PCT Publication WO 2014/055897, for example, Ab-14, Ab-16, Ab-30, Ab-31, Ab-42, Ab-50, Ab-52, or Ab-55, or an antibody that contains the CDR regions of any of these antibodies.

In some embodiments, the CTLA-4 antagonist is an antibody that specifically binds CTLA-4. In some embodiments, the antibody that binds CTLA-4 is ipilimumab (YERVOY) or tremelimumab (CP-675,206). In some embodiments, the CTLA-4 antagonist a CTLA-4 fusion protein, for example, KAHR-102.

In some embodiments, the LAG3 antagonist is an antibody that specifically binds LAG3. In some embodiments, the antibody that binds LAG3 is IMP701, IMP731, BMS-986016, LAG525, and GSK2831781. In some embodiments, the LAG3 antagonist includes a soluble LAG3 receptor, for example, IMP321.

In some embodiments, the KIR antagonist is an antibody that specifically binds KIR. In some embodiments, the antibody that binds KIR is lirilumab.

In some embodiments, an immune response stimulating agent is selected from the group consisting of: a CD28 agonist, a 4-1BB agonist, an OX40 agonist, a CD27 agonist, a CD80 agonist, a CD86 agonist, a CD40 agonist, and a GITR agonist.

In some embodiments, the OX40 agonist includes OX40 ligand, or an OX40-binding portion thereof. For example, the OX40 agonist may be MEDI6383. In some embodiments, the OX40 agonist is an antibody that specifically binds OX40. In some embodiments, the antibody that binds OX40 is MEDI6469, MEDI0562, or MOXR0916 (RG7888). In some embodiments, the OX40 agonist is a vector (e.g., an expression vector or virus, such as an adenovirus) capable of expressing OX40 ligand. In some embodiments the OX40-expressing vector is Delta-24-RGDOX or DNX2401.

In some embodiments, the 4-1BB (CD137) agonist is a binding molecule, such as an anticalin. In some embodiments, the anticalin is PRS-343. In some embodiments, the 4-1BB agonist is an antibody that specifically binds 4-1BB. In some embodiments, antibody that binds 4-1BB is PF-2566 (PF-05082566) or urelumab (BMS-663513).

In some embodiments, the CD27 agonist is an antibody that specifically binds CD27. In some embodiments, the antibody that binds CD27 is varlilumab (CDX-1127).

In some embodiments, the GITR agonist comprises GITR ligand or a GITR-binding portion thereof. In some embodiments, the GITR agonist is an antibody that specifically binds GITR. In some embodiments, the antibody that binds GITR is TRX518, MK-4166, or INBRX-110.

In some embodiments, immune response stimulating agents include, but are not limited to, cytokines such as chemokines, interferons, interleukins, lymphokines, and members of the tumor necrosis factor (TNF) family. In some embodiments, immune response stimulating agents include immunostimulatory oligonucleotides, such as CpG dinucleotides.

In some embodiments, an immune response stimulating agent includes, but is not limited to, anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-PD-L2 antibodies, anti-CTLA-4 antibodies, anti-CD28 antibodies, anti-CD80 antibodies, anti-CD86 antibodies, anti-4-1BB antibodies, anti-OX40 antibodies, anti-KIR antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-CD27 antibodies, anti-CD40 antibodies, anti-GITR antibodies, anti-TIGIT antibodies, anti-CD20 antibodies, anti-CD96 antibodies, or anti-IDO1 antibodies.

In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding polypeptide or agent described herein in combination with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is a hematologic cancer.

In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a GITR-binding polypeptide or agent described herein in combination with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is breast cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is a hematologic cancer.

In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of an OX40-binding polypeptide or agent described herein in combination with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is a hematologic cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is breast cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is a hematologic cancer.

In some embodiments, a method of treating cancer in a subject comprises administering to the subject a therapeutically effective amount of a CD40-binding polypeptide or agent described herein in combination with a checkpoint inhibitor. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-1 antibody and the cancer is a hematologic cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is melanoma. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is lung cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is bladder cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is breast cancer. In some embodiments, the checkpoint inhibitor is an anti-PD-L1 antibody and the cancer is a hematologic cancer.

In certain embodiments of the methods described herein, the treatment involves the administration of a polypeptide or agent of the present invention in combination with radiation therapy. Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of radiation therapy. Dosing schedules for such radiation therapy can be determined by the skilled medical practitioner.

In certain embodiments of the methods described herein, the treatment involves the administration of a polypeptide or agent of the present invention in combination with anti-viral therapy. Treatment with a polypeptide or agent can occur prior to, concurrently with, or subsequent to administration of antiviral therapy. The anti-viral drug used in combination therapy will depend upon the virus the subject is infected with.

Combined administration can include co-administration, either in a single pharmaceutical formulation or using separate formulations, or consecutive administration in either order but generally within a time period such that all active agents can exert their biological activities simultaneously.

It will be appreciated that the combination of a polypeptide or agent described herein and at least one additional therapeutic agent may be administered in any order or concurrently. In some embodiments, the polypeptide or agent will be administered to patients that have previously undergone treatment with a second therapeutic agent. In certain other embodiments, the polypeptide or agent and a second therapeutic agent will be administered substantially simultaneously or concurrently. For example, a subject may be given a polypeptide or agent while undergoing a course of treatment with a second therapeutic agent (e.g., chemotherapy). In certain embodiments, a polypeptide or agent will be administered within 1 year of the treatment with a second therapeutic agent. In certain alternative embodiments, a polypeptide or agent will be administered within 10, 8, 6, 4, or 2 months of any treatment with a second therapeutic agent. In certain other embodiments, a polypeptide or agent will be administered within 4, 3, 2, or 1 weeks of any treatment with a second therapeutic agent. In some embodiments, a polypeptide or agent will be administered within 5, 4, 3, 2, or 1 days of any treatment with a second therapeutic agent. It will further be appreciated that the two (or more) agents or treatments may be administered to the subject within a matter of hours or minutes (i.e., substantially simultaneously).

For the treatment of a disease, the appropriate dosage of a polypeptide or agent of the present invention depends on the type of disease to be treated, the severity and course of the disease, the responsiveness of the disease, whether the polypeptide or agent is administered for therapeutic or preventative purposes, previous therapy, the patient's clinical history, and so on, all at the discretion of the treating physician. The polypeptide or agent can be administered one time or over a series of treatments lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved (e.g., reduction in tumor size). Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient and will vary depending on the relative potency of an individual agent. The administering physician can determine optimum dosages, dosing methodologies, and repetition rates. In certain embodiments, dosage is from 0.01 μg to 100 mg/kg of body weight, from 0.1 μg to 100 mg/kg of body weight, from 1 μg to 100 mg/kg of body weight, from 1 mg to 100 mg/kg of body weight, 1 mg to 80 mg/kg of body weight from 10 mg to 100 mg/kg of body weight, from 10 mg to 75 mg/kg of body weight, or from 10 mg to 50 mg/kg of body weight. In certain embodiments, the dosage of the polypeptide or agent is from about 0.1 mg to about 20 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.1 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.25 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 0.5 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 1 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 1.5 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 2 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 2.5 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 5 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 7.5 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 10 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 12.5 mg/kg of body weight. In some embodiments, the dosage of the polypeptide or agent is about 15 mg/kg of body weight. In certain embodiments, the dosage can be given once or more daily, weekly, monthly, or yearly. In certain embodiments, the polypeptide or agent is given once every week, once every two weeks, once every three weeks, or once every four weeks.

In some embodiments, a polypeptide or agent may be administered at an initial higher “loading” dose, followed by one or more lower doses. In some embodiments, the frequency of administration may also change. In some embodiments, a dosing regimen may comprise administering an initial dose, followed by additional doses (or “maintenance” doses) once a week, once every two weeks, once every three weeks, or once every month. For example, a dosing regimen may comprise administering an initial loading dose, followed by a weekly maintenance dose of, for example, one-half of the initial dose. Or a dosing regimen may comprise administering an initial loading dose, followed by maintenance doses of, for example one-half of the initial dose every other week. Or a dosing regimen may comprise administering three initial doses for 3 weeks, followed by maintenance doses of, for example, the same amount every other week.

As is known to those of skill in the art, administration of any therapeutic agent may lead to side effects and/or toxicities. In some cases, the side effects and/or toxicities are so severe as to preclude administration of the particular agent at a therapeutically effective dose. In some cases, drug therapy must be discontinued, and other agents may be tried. However, many agents in the same therapeutic class often display similar side effects and/or toxicities, meaning that the patient either has to stop therapy, or if possible, suffer from the unpleasant side effects associated with the therapeutic agent.

In some embodiments, the dosing schedule may be limited to a specific number of administrations or “cycles”. In some embodiments, the polypeptide or agent is administered for 3, 4, 5, 6, 7, 8, or more cycles. For example, the polypeptide or agent is administered every 2 weeks for 6 cycles, the polypeptide or agent is administered every 3 weeks for 6 cycles, the polypeptide or agent is administered every 2 weeks for 4 cycles, the polypeptide or agent is administered every 3 weeks for 4 cycles, etc. Dosing schedules can be decided upon and subsequently modified by those skilled in the art.

Thus, the present invention provides methods of administering to a subject the polypeptides or agents described herein comprising using an intermittent dosing strategy for administering one or more agents, which may reduce side effects and/or toxicities associated with administration of a polypeptide or agent, chemotherapeutic agent, etc. In some embodiments, a method for treating cancer in a human subject comprises administering to the subject a therapeutically effective dose of a polypeptide or agent in combination with a therapeutically effective dose of a chemotherapeutic agent, wherein one or both of the agents are administered according to an intermittent dosing strategy. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 2 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 3 weeks. In some embodiments, the intermittent dosing strategy comprises administering an initial dose of a polypeptide or agent to the subject, and administering subsequent doses of the polypeptide or agent about once every 4 weeks. In some embodiments, the polypeptide or agent is administered using an intermittent dosing strategy and the chemotherapeutic agent is administered weekly.

V. SCREENING

The present invention provides screening methods to identify agents that modulate the immune response. In some embodiments, the present invention provides methods for screening candidate agents, including but not limited to, proteins, antibodies, peptides, peptidomimetics, small molecules, compounds, or other drugs, which modulate the immune response.

In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent has an effect on immune response cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of increasing the activity of immune cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of increasing the activity of cytolytic cells, such as CTLs and/or NK cells. In some embodiments, a method of screening for a candidate agent that modulates the immune response comprises determining if the polypeptide or agent is capable of inhibiting the activity of suppressor cells, such as Tregs and/or MDSCs.

VI. KITS COMPRISING AGENTS DESCRIBED HEREIN

The present invention provides kits that comprise the polypeptides or agents described herein and that can be used to perform the methods described herein. In certain embodiments, a kit comprises at least one purified agent in one or more containers. In some embodiments, the kits contain all of the components necessary and/or sufficient to perform a detection assay, including all controls, directions for performing assays, and any necessary software for analysis and presentation of results. One skilled in the art will readily recognize that the disclosed agents of the present invention can be readily incorporated into one of the established kit formats which are well known in the art.

Further provided are kits that comprise a polypeptide or agent as well as at least one additional therapeutic agent. In certain embodiments, the second (or more) therapeutic agent is a chemotherapeutic agent. In certain embodiments, the second (or more) therapeutic agent is an angiogenesis inhibitor.

Embodiments of the present disclosure can be further defined by reference to the following non-limiting examples, which describe in detail preparation of certain antibodies of the present disclosure and methods for using antibodies of the present disclosure. It will be apparent to those skilled in the art that many modifications, both to materials and methods, may be practiced without departing from the scope of the present disclosure.

EXAMPLES Example 1 Generation of Single Chain GITRL Trimer Constructs

As discussed herein hGITRL proteins organize into a homotrimer at the surface of cells and interact and/or engage with three GITR molecules. A representative diagram of a GITRL trimer on a cell surface is shown in FIG. 1A. The crystal structure of a human GITRL trimer was examined by the inventors and it was observed that the N-terminal amino acid residues from one monomer and the C-terminal amino acid residues from a second monomer were in close proximity to each other. This suggested that a very short span of amino acid residues, for example only 3-7 residues, might be sufficient to bridge the distance between each monomer and thereby enable a single chain GITRL trimer to be produced. Upon further analysis of the structure and sequence of GITRL, it was observed that there exists a span of several amino acids between the transmembrane domain and the TNF homology domain of the GITRL protein, this span is referred to as the “stalk” region (see FIGS. 1A and 1B). The inventors hypothesized that it would be possible to utilize this short stalk region to bridge the distance from the C-terminus of a GITRL monomer to the N-terminus of an adjacent GITRL monomer and in this fashion construct a single chain GITRL trimer that was devoid of exogenous peptide linker sequences.

A membrane-bound single chain hGITRL trimer construct was generated using the transmembrane region (signal-anchor sequence—amino acids 51-70 of SEQ ID NO:1) and three copies of the stalk region and TNF family domain (amino acids 71-199 of SEQ ID NO:1; also SEQ ID NO:3). The sequence encompassing the stalk region and the TNF family domain is also referred to herein as the extracellular domain of GITRL. A representative diagram is shown in FIG. 1D.

A soluble single chain hGITRL trimer construct was also generated (FIG. 1E). An additional construct was generated that comprised three copies of the extracellular domain of human GITRL linked to a human IgG1 Fc region (336B11; SEQ ID NO:6 with signal sequence and SEQ ID NO:7 without signal sequence). A representative diagram is shown in FIG. 1F.

The general outline disclosed herein for generating the GITRL trimer construct can be followed to produce other TNFSF trimer constructs. For example, the inventors have generated several OX40L trimers (SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:71; and SEQ ID NO:72), OX40L trimer-Fc proteins (SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, and SEQ ID NO:48), CD40L trimers (SEQ ID NO:85 and SEQ ID NO:88), and CD40L trimer-Fc proteins (SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91 and SEQ ID NO:92).

Example 2 FACS Analysis of GITRL Trimer Binding to GITR

To test the ability of a single chain hGITRL trimer to bind human GITR, binding studies were conducted. Human HEK-293 cells were transiently co-transfected with expression vectors encoding (1) the membrane bound single chain hGITRL trimer construct described above and (2) green fluorescent protein (GFP) as a transfection marker. Twenty-four hours post-transfection, the cells were incubated with hGITR-Fc or a control fusion protein. The cells were stained with a PE-conjugated anti-human Fc secondary antibody and analyzed by flow cytometry.

As shown in FIG. 2, hGITR-Fc was able to bind to the single chain hGITRL trimer on the cell surface. These results demonstrated that the single chain hGITRL construct formed into a biologically functional trimeric structure and was able to interact with hGITR.

Example 3 GITRL Trimer Fusion Proteins and Binding

The inventors hypothesized that a soluble single chain GITRL trimer could be generated using an immunoglobulin Fc region backbone and that the GITRL trimer could potentially be linked to the N-terminus of the Fc region or the C-terminus of the Fc region. This would generate a molecule containing two GITRL trimers and could offer increased activity. The structural flexibility of the Fc region backbone could also allow generation of bispecific homodimeric agents. For example, an antibody-GITRL fusion protein could be designed wherein the single chain GITRL trimer is linked to an immunoglobulin heavy chain To form an antigen binding site, the immunoglobulin heavy chain could be associated with an immunoglobulin light chain. An antibody-GITRL fusion protein could comprise an antibody heavy chain and a light chain as part of a single chain immunoglobulin. In a different design, an antibody-GITRL fusion protein could comprise a heavy chain variable region and a light chain variable region, such as a Fab or scFv.

Alternatively, one could design bispecific heterodimeric agents wherein one arm comprises a Fc fusion protein that comprises a single chain GITRL trimer and a second arm comprises a second Fc fusion protein that comprises an immunoglobulin heavy chain. As described above, to form an antigen binding site, the immunoglobulin heavy chain could be associated with an immunoglobulin light chain. The second arm could comprise an antibody heavy chain and a light chain as part of a single chain immunoglobulin or a Fc region linked to heavy chain variable region and a light chain variable region, such as a Fab or scFv. Schematic representations of some of these formats are shown in FIG. 3.

Several of these formats were produced as recombinant proteins using mouse IgG1 or IgG2 Fc regions, human IgG1 or IgG2 Fc regions, and mouse IgG1 or IgG2 antibodies. A pair of human IgG2 CH3 domain variants that preferentially heterodimerize were used to produce bispecific heterodimeric agents. 336B3 is a single chain mouse GITRL trimer linked to the C-terminus of a mouse IgG2a Fc region. 336B10 is a single chain mouse GITRL trimer linked to the N-terminus of a mouse IgG2a Fc region. 336B11 is a single chain human GITRL trimer linked to the N-terminus of a human IgG1 region. 336B11 comprises a fusion protein encoded by the plasmid “hGITRL-hIgG1” deposited with ATCC at 10801 University Boulevard, Manassas, Va., USA, under the conditions of the Budapest Treaty on Apr. 21, 2015 and assigned designation number PTA-122112. 336B1 is a single chain mouse GITRL trimer linked to a mouse IgG1 antibody. 336B2 is a single chain mouse GITRL trimer linked to a mouse IgG2a antibody. 336B4 is a heterodimeric agent wherein one arm is a mouse GITRL trimer linked to a human IgG2 Fc region and the second arm is a mouse antibody variable region linked to a human IgG2 Fc region.

To test the ability of these fusion proteins to interact with GITR, binding studies were conducted. Human HEK-293 cells were transiently co-transfected with expression vectors encoding (1) a full-length mouse GITR (cell surface expression) and (2) GFP as a transfection marker. Twenty-four hours post-transfection, the cells were incubated with the GITRL fusion proteins or control proteins. The cells were stained with a PE-conjugated anti-human or anti-mouse Fc secondary antibody and analyzed by flow cytometry.

As shown in FIG. 4, Fc fusion proteins comprising GITRL trimer(s) strongly bind to GITR expressed at the cell surface. The results demonstrated that the GITRL trimer could bind GITR when linked at either the N-terminus or the C-terminus of the Fc protein and that GITRL could bind GITR when expressed as a single trimer or when multiple trimers were present. In addition, the GITRL trimers were functional when fused to Fc regions or antibodies of different isotypes. Importantly, the GITRL trimer also bound GITR in the context of a bispecific homodimeric or heterodimeric molecule.

A comparison of a single chain mGITRL trimer-Fc (336B10) and a single chain hGITRL trimer-Fe (336B11) to bind mouse GITR and human GITR, respectively, was undertaken. Human HEK-293 cells were transiently co-transfected with expression vectors encoding (1) a full-length mouse or human GITR (cell surface expression) and (2) GFP as a transfection marker. Twenty-four hours post-transfection, the cells were incubated with mGITRL trimer-Fc 336B10 or hGITRL trimer-Fc 336B11 over a range of concentrations (2-fold dilutions 20 m/ml to 0.156 m/ml). The cells were stained with a PE-conjugated anti-human or anti-mouse Fc secondary antibody and analyzed by flow cytometry.

As shown in FIG. 5, single chain mGITRL trimer-Fc and single chain hGITRL trimer-Fc strongly bind to their respective GITRs and show similar dose responses for binding.

Additional studies have shown that human GITRL trimer-Fc 336B11 binds cynomolgus monkey GITR.

Example 4 Activation of GITR Signaling by GITRL Trimer-Fc

To determine whether the single chain GITRL trimer-Fc proteins would induce signaling through GITR, luciferase reporter assays were conducted. A HEK-293 cell line which contains a stably transfected NF-kB-luciferase reporter gene and mouse GITR cDNA was used. Cells were plated into a 96 well plate and incubated with single chain mGITRL-Fc fusion protein 336B3, single chain mGITRL-Fc fusion protein 336B6, anti-GITR antibody DTA-1, or a control antibody over a range of concentrations (5-fold dilutions 20 m/ml to 0.01 m/ml). 336B3 is a homodimer of the mGITRL trimer-Fc with the trimer linked at the C-terminus of an IgG2A Fc region (“two-trimer” version). 336B6 has only one of the Fc regions linked to a GITRL trimer linked at the N-terminus of an IgG2A Fc region (a “one-armed” or “one-trimer” version). DTA-1 is an agonist antibody targeting GITR and was included as a positive control.

As shown in FIG. 6, a Fc fusion protein containing a single GITRL trimer (336B6) and a Fc fusion protein containing two GITRL trimers (336B3) were able to robustly stimulate luciferase. These results suggest that the GITRL trimers are able to strongly induce GITR signaling in a biologically relevant manner. Interestingly, the single chain GITRL trimer formats were able to induce more robust GITR signaling than the agonist anti-GITR antibody. Furthermore, in this study a fusion protein containing two copies of the single chain GITRL trimer (336B3) was able to provide a higher maximal stimulation than a fusion protein containing only one GITRL trimer (336B6). These results are consistent with our hypothesis that the single chain GITRL trimer format could be more potent than an agonist antibody format for achieving activation of GITR and that multiple copies of GITRL trimers could function as a “supercluster” to achieve even more potent GITR activation.

These experiments were repeated with a human GITRL trimer-Fc fusion protein (336B11) and HEK-293 cells stably transfected with the NF-kB-luciferase reporter gene and human GITR cDNA. Cells were plated into a 96 well plate and incubated with single chain hGITRL-Fc fusion protein 336B11 over a range of concentrations (3-fold dilutions 20 μg/ml to 0.08 μg/ml).

As shown in FIG. 7, the single chain human GITRL trimer-Fc protein 336B11 protein was able to robustly stimulate luciferase. These results are comparable to the results obtained with murine GITRL trimers and indicate that the human GITRL trimer is able to stimulate GITR signaling.

Example 5 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein

The murine colon tumor line CT26.WT was implanted subcutaneously (25,000 cells/mouse) in Balb/c mice. Mice were treated with 0.25 mg/mouse of single chain mGITRL trimer-Fc 336B3, agonist anti-GITR antibody DTA-1, or a control antibody (n=10 per group). Mice were dosed by intraperitoneal injection on days 7, 10, 14, and 17. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

As is shown in FIG. 8A, treatment with mGITRL trimer-Fc 336B3 strongly inhibited and/or prevented growth of the CT26.WT tumors. Treatment with the agonist anti-GITR antibody DTA-1 also inhibited tumor growth. A more nuanced picture of the difference between 336B3 and DTA-1 can be seen by looking at the results from the individual mice within each group. As shown in FIG. 8B, treatment with 336B3 inhibited tumor growth in all ten mice while it appeared that treatment with DTA-1 inhibited tumor growth to a lesser extent and failed to block tumor growth in one of ten mice. These results indicate that the single chain GITRL trimer is active as an immunotherapeutic agent and could be more potent in achieving suppression of tumor growth than an agonist GITR antibody.

Example 6 ELISpot Assay for IFN-Gamma and IL-10

ELISpot is a highly sensitive immunoassay for the detection of cytokine-secreting cells. Briefly, an ELISpot assay employs a capture antibody specific for a desired cytokine, pre-coated onto the wells of a microplate. Cells are dispensed into the wells and the immobilized antibody in the immediate vicinity of any cytokine-secreting cell binds the secreted cytokine. Standard wash steps and incubation with appropriate detection reagents follow. For example, a biotinylated detection antibody followed by streptavidin conjugated to alkaline-phosphatase and a colored substrate solution are commonly used. A colored precipitate forms at the sites of cytokine localization and appears as a spot, with each individual spot representing an individual cytokine-secreting cell. The spots may be counted with an automated reader system or manually using a microscope.

Interferon (IFN)-gamma secreting cells were detected using a mouse IFN-gamma ELISpot kit (MabTech, Cincinnati, Ohio). Cells were isolated from the spleens of CT26.WT tumor-bearing mice treated with mGITRL trimer-Fc 336B, anti-mGITR antibody DTA-1, or a control, as described above in Example 5. Splenocytes from each mouse (2×10⁵ cells/well) were dispensed into the provided plates, which were pre-coated with a capture antibody specific for murine IFN-gamma. The cells were cultured in the presence or the absence of a tumor specific CD8⁺ T-cell peptide (AH-1) and incubated at 37° C. The sequence of the AH-1 peptide (SPSYVYHQF; SEQ ID NO:54) is the H2-L^(d)-restricted epitope (amino acids 6-14) of the gp70 envelope protein of an ecotropic murine leukemia provirus endogenous to the CT26.WT cell line. After 48 hours, cells secreting IFN-gamma were detected following the manufacturer's instructions. Spots were counted using a 6000 F-z Bioreader (Biosys, Miami, Fla.). Data are expressed as the mean±S.E.M spots/well.

As shown in FIG. 9A, tumor-specific IFN-gamma-secreting CD8⁺ T-cells were increased in mice treated with mGITRL trimer-Fc 336B3 and with anti-mGITR antibody DTA-1 when cells were incubated with the AH-1 peptide. The increase was greater with 336B3, approximately 2.5-fold increase versus an approximate 1.9-fold increase with DTA-1. Furthermore, in the mice treated with 336B3 the number of IFN-gamma secreting T-cells was increased even in the absence of a tumor-specific peptide. This increase was not seen with the anti-GITR antibody DTA-1.

IFN-gamma is generally produced by NK cells, Th1 CD4+ T-cells, CD8+ T-cells, antigen presenting cells, and B-cells. Studies have suggested a role for IFN-gamma in tumor immunity and that it may be a regulator of anti-tumor activity mediated by other cytokines, in particular IL-12 and IL-2. Thus, treatment with a GITRL trimer-Fc that results in an increase in IFN-gamma should enhance anti-tumor immunity.

IL-10 secreting cells were detected using a mouse IL-10 ELISPOT kit (MabTech). Cells were isolated from the spleens of CT26.WT tumor-bearing mice treated with mGITRL trimer-Fc 336B, anti-mGITR antibody DTA-1, or a control. Splenocytes (5×10⁵/well) from each mouse within each treatment group were dispensed into a 96-well plate coated with an antibody specific for mouse IL-10. The cells were cultured in the presence or the absence of a tumor specific CD8⁺ T-cell peptide (AH-1) and incubated at 37° C. After 48 hours cells secreting IL-10 were detected following the manufacturer's instructions. Images were captured using a Bioreader 6000 F-z instrument (BioSys) and spot number, spot area, and/or total optical density were determined. Data are expressed as the mean±S.E.M spots/well.

As shown in FIG. 9B, IL-10 secreting cells were significantly increased in mice treated with anti-mGITR antibody DTA-1 as compared to mice treated with a control whether the cells were incubated in the presence or absence of the tumor specific peptide. Interestingly, the number of IL-10 secreting cells from mice treated with mGITRL trimer-Fc 336B3 was significantly increased only when the tumor-specific peptide was not present, demonstrating only a small increase as compared to mice treated with control when the cells were stimulated with the tumor-specific peptide AH-1.

IL-10 is generally produced by Tregs and helper T-cells. IL-10 was originally recognized as a Th2 cytokine that modulates growth and/or differentiation of innate immune cells and suppresses the activation and effector functions of T-cells, particularly cytotoxic T-cells. More recently, IL-10 has been shown to have some immune stimulatory effects and thus is viewed as having pleiotropic functions. Since Th1 cytokines such as IFN-gamma counter-regulate production of Th2 cytokines including IL-10, 336B3 treatment may suppress IL-10 production by inducing significant production of IFN-gamma. Thus, in conjunction with increased IFN-gamma production, 336B3 treatment may promote anti-tumor immunity by suppressing IL-10 production.

These data suggest that the GITRL trimer-Fc can promote tumor-specific CD8⁺ T-cell activity by significantly producing IFN-gamma. These results may also suggest that the GITRL trimer-Fc can more efficiently induce anti-tumor immunity than an agonist anti-GITR antibody. Furthermore, these results may suggest that the mechanism by which the GITRL trimer-Fc is affecting immune cells and/or immune responses is different than the mechanism of the anti-GITR antibody.

Example 7 Cell Cytotoxicity Assay

Natural killer cells or NK cells are a type of cytotoxic lymphocyte critical to the innate immune system. NK cell activity in mice treated with mGITRL trimer-Fc 336B3 was assessed by measuring the cytotoxic activity of cells on tumor targets. Cells were harvested from the spleens of the CT26.WT tumor-bearing mice described above in Example 5. Cells were plated in 96-well V-bottom plates in RPMI 1640 culture medium (Gibco/Life Technologies, Grand Island, N.Y.) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). CT26.WT target cells were labeled with 10 μM calcein AM (Life Technologies) for 1 hour at 37° C. and then combined with the splenocytes at an effector:target (E:T) ratio of 25:1. Following a 4 hour incubation at 37° C., cell-free supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. The percentage of specific cell lysis was determined as: % lysis=100×(ER−SR)/(MR−SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.

As shown in FIG. 10, NK cells from CT26.WT tumor-bearing mice demonstrated an increased ability to kill CT26.WT target cells when the mice had been treated with mGITRL trimer-Fc 336B3 as compared to cells from mice treated with control antibody. This effect was greater than any effect seen with the anti-mGITR antibody DTA-1. Although the amount of lysis was low, these results suggest that treatment with the mGITRL trimer-Fc can increase NK activity and enhance anti-tumor immune responses. Furthermore, the effect with mGITRL trimer-Fc was more potent that what was observed with an anti-GITR antibody.

Example 8 Regulatory T-Cell (Treg) Assay

Regulatory T-cells (Tregs) play an essential role in the maintenance of homeostasis and prevention of autoimmune responses. Tregs are a small subset of T-cells, most which are CD4+ cells and express CD25 (an IL-2 receptor alpha chain) and other Treg cell-related molecular markers. Foxp3, a transcription factor, has been recognized to be a factor for Treg cell development and function. Foxp3 has also been considered as a specific marker to define and identify Treg cells from other T cell subpopulations although this has been challenged on its specificity in human Treg cells. In addition to CD4+Treg cells, CD8+Treg cells represent another cell population and Foxp3 may not be so crucial for their development and function when compared to CD4+Treg cells.

The functionality of Tregs in mice treated with mGITRL trimer-Fc 336B3 was evaluated by determining the effect Tregs had on proliferation of naïve CD4+ or CD8+ T-cells. Naïve T-cells were purified from the spleens of untreated mice using a mouse CD3+ T-cell enrichment column (R&D Systems). These purified T-cells were labeled with 5 μM violet tracking dye (VTD; Life Technologies). 2×10⁵ VTD-labeled T-cells were incubated with anti-CD3 and anti-CD28 antibody-coated beads to stimulate cell proliferation. Tregs were isolated from the spleens of CT26.WT tumor-bearing mice (see Example 5) treated with mGITRL trimer-Fc 336B3, anti-mGITR antibody DTA-1, or control using a mouse Treg isolation kit (Miltenyi Biotec). To determine the impact of Tregs on T-cell proliferation, the stimulated VTD-labeled T-cells (effectors) were co-cultured with the isolated splenic Tregs (Effector:Treg ratio of 1:0.5). On day 4, cells were washed, and incubated with anti-mouse CD4 or anti-mouse CD8 antibodies. Cells were evaluated by FACS analysis using a BD FACSCanto II instrument and BD FACSDiva software v6.1.3. VTD signals are reduced by half as the labeled cells divide, therefore the analysis gate was set between the maximum signal obtained with no Treg cells in the assay and the minimum signal obtained with no anti-CD3/CD28 stimulation. The percentage of cells within this region (reduced VTD expression) on CD4+ T-cells and CD8+ T-cells was used to calculate CD4+ and CD8+ T-cell proliferation. Percent suppression was calculated as [maximum signal−(sample signal/maximum signal)]×100.

As shown in FIG. 11, treatment with mGITRL trimer-Fc 336B3 strongly decreased the suppressive function of Tregs on naïve CD4+ T-cell proliferation as compared to suppression seen with Tregs from mice treated with anti-mGITR antibody DTA-1 or control. Similarly, treatment with 336B3 reduced the suppressive function of Tregs on naïve CD8+ T-cell proliferation as compared to suppression seen with Tregs from mice treated with DTA-1 or control.

These results suggest that treatment with mGITRL trimer-Fc can lead to reduced Treg function and/or suppression. This effect can be thought of as “taking the brake off” the immune response. Thus, a reduction of Treg function could enhance total anti-tumor immune responses.

Example 9 Myeloid-Derived Suppressor Cell (MDSC) Assay

Studies have identified myeloid-origin cells that are potent suppressors of tumor immunity and therefore a significant impediment to cancer immunotherapy (see, for example, Ostrand-Rosenberg et al., 2009, J. Immunol., 182:4499-4506). Myeloid-derived suppressor cells (MDSCs) accumulate in the blood, lymph nodes, bone marrow, and at tumor sites in most patients and experimental animals with cancer. MDSCs have been shown to inhibit both adaptive and innate immunity.

It is believed that MDSCs facilitate cancer progression by inhibiting anti-tumor immune responses, promoting angiogenesis, and creating a pre-metastatic environment. MDSCs suppress the proliferation and activation of CD4⁺ T-cells and CD8⁺ T-cells, thereby inhibiting anti-tumor immunity. Importantly, MDSCs facilitate the generation of Tregs.

MDSCs are a heterogeneous family of myeloid cells. In mice, MDSCs are characterized by the cell surface expression of the myeloid lineage differentiation antigens Gr1 and CD11b. MDSCs can be divided into two subpopulations: granulocytic MDSCs (G-MDSC) and monocytic MDSCs (M-MDSC). G-MDSCs typically have multi-lobed nuclei and a CD11b⁺Ly6G⁺Ly6C^(low) phenotype, whereas M-MDSCs have a monocytic morphology and a CD11b⁺Ly6G^(+/−)Ly6C^(high) phenotype. Both populations of MDSCs have been shown to suppress T-cell responses by multiple mechanisms including increased production of arginase, inducible nitric oxide synthase (iNOS), nitric oxide, and reactive oxygen species. Thus, MDSCs contribute to an immunosuppressive tumor microenvironment and may limit the effects of anti-tumor immune responses.

The functionality of MDSCs in mice treated with mGITRL trimer-Fc 336B3 was evaluated by determining the effect MDSCs had on proliferation of naïve CD4+ or CD8+ T-cells. Naïve T-cells were purified from the spleens of untreated mice using a mouse CD3+ T-cell enrichment column (R&D Systems). These purified T-cells were labeled with 5 μM violet tracking dye (VTD; Life Technologies). 2×10⁵ VTD-labeled T-cells were incubated with anti-CD3 and anti-CD28 antibody-coated beads to stimulate cell proliferation. MDSCs were isolated from the spleens of CT26.WT tumor-bearing mice (see Example 5) treated with mGITRL trimer-Fc 336B3, anti-mGITR antibody DTA-1, or control using a mouse MDSC isolation kit (Miltenyi Biotec). To determine the impact of MDSC on T-cell proliferation, the stimulated VTD-labeled T-cells (effectors) were co-cultured with the isolated splenic MDSCs (effector:MDSC ratio of 1:1). On day 4, cells were washed, and incubated with anti-mouse CD4 or anti-mouse CD8 antibodies. Cells were evaluated by FACS analysis using a BD FACSCanto II instrument and BD FACSDiva software v6.1.3. VTD signals are reduced by half as the labeled cells divide, therefore the analysis gate was set between the maximum signal obtained with no MDSCs in the assay and the minimum signal obtained with no anti-CD3/CD28 stimulation. The percentage of cells within this region (reduced VTD expression) on CD4+ T-cells and CD8+ T-cells was used to calculate CD4+ and CD8+ T-cell proliferation. Percent suppression was calculated as [maximum signal−(sample signal/maximum signal)]×100.

As shown in FIG. 12, treatment with mGITRL trimer-Fc 336B3 strongly decreased the suppressive function of MDSCs on naïve CD4+ T-cell proliferation as compared to suppression seen with MDSCs from mice treated with control. The reduction was less pronounced in cells from mice treated with the anti-GITR antibody. In contrast, treatment with 336B3 had only a slight effect on the suppressive function of MDSCs on naïve CD8+ T-cell proliferation as compared to suppression seen with MDSCs from mice treated with control.

These results suggest that treatment with mGITRL trimer-Fc may have some effect on MDSC function and/or suppression. A reduction of MDSC function could further enhance total anti-tumor immune responses.

Example 10 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein

Renca is a Balb/c-derived renal adenocarcinoma cell line obtained from ATCC. Renca cells were implanted subcutaneously (5×10⁵ cells/mouse) in Balb/c mice and allowed to grow for seven days reaching an average size of approximately 78 mm³. Mice were treated with 0.25 mg/mouse of single chain mGITRL trimer-Fc 336B3, agonist anti-GITR antibody DTA-1, or a control antibody (n=10 per group). Mice were dosed by intraperitoneal injection on days 7, 11, and 14. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

As is shown in FIG. 13, treatment with mGITRL trimer-Fc 336B3 strongly inhibited growth of the Renca tumors. Treatment with the agonist anti-GITR antibody DTA-1 also inhibited tumor growth but to a lesser extent than 336B3. When results were assessed at an individual mouse level, tumors regressed to a size smaller than the size at the first treatment in 9 of 20 mice (45%) treated with 336B3, with three mice having undetectable tumors by Day 25. In contrast, tumors regressed to a size smaller than the size at the first treatment in only 5 of 20 mice (25%) treated with DTA-1, with four mice having undetectable tumors by Day 25.

These results support the idea that the single chain GITRL trimer is active as an immuno-oncologic agent in tumors of different origin and that it may be more potent in achieving inhibition of tumor growth than an agonist GITR antibody.

Example 11 Cell Cytotoxicity Assays

For natural killer (NK) cytotoxicity assays, the mouse lymphoblast cell line YAC-1 was used. Cells were cultured in RPMI 1640 culture medium (Gibco/Life Technologies, Grand Island, N.Y.) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) at 37° C. in a humidified atmosphere of 5% CO2. YAC-1 cells are known to be sensitive to NK cell activity and are a good target for NK cell assays.

Cells were harvested from the spleens of the mice described above in Example 10. Cells were plated in 96-well V-bottom plates in RPMI 1640 culture medium (Gibco/Life Technologies, Grand Island, N.Y.) supplemented with 10% (v/v) fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco). YAC-1 target cells were labeled with 10 μM calcein AM (Life Technologies) for 1 hour at 37° C. and then combined with the splenocytes at an effector:target ratio of 25:1 or 50:1. Following a 4 hour incubation at 37° C., cell-free supernatants were harvested and calcein release was quantified on a fluorometer at an excitation of 485 nm and an emission of 535 nm. The percentage of specific cell lysis was determined as: % lysis=100×(ER−SR)/(MR−SR), where ER, SR, and MR represent experimental, spontaneous, and maximum calcein release, respectively. Spontaneous release is the fluorescence emitted by target cells incubated in media alone (i.e., in the absence of effector cells), while maximum release is determined by lysing target cells with an equal volume of 10% SDS.

NK cells from Renca cell-injected mice demonstrated an increased ability to kill YAC-1 target cells when the mice had been treated with mGITRL trimer-Fc 336B3 as compared to cells from mice treated with control. Treatment with anti-mGITR antibody DTA-1 also had an increased ability to kill target cells but to a smaller extent than 336B3 (FIG. 14A).

A CD8⁺ T-cell specific MHC class I tumor peptide sequence is not known for the Renca cell line, therefore the Renca cells were used as stimulators. Renca cells were treated with 25 μg/ml mitomycin C (Sigma-Aldrich) for 30 minutes at 37° C., washed, and resuspended at 10⁷ cells/ml in RPMI-1640 media containing 10% FCS, 2 mM L-glutamine, and antibiotics. Splenocytes were co-cultured with the mitomycin-treated Renca cells in the presence of IL-2 (2 ng/ml), incubated for 5 days at 37° C., harvested, counted, and used in cytotoxicity assays as described above. Calcein AM-labeled Renca cells were used as targets at an effector:target ratio of 25:1. Calcein release was determined after 4 hours and specific lysis was calculated as described above.

As shown in FIG. 14B, CD8⁺ cytotoxic cells from Renca cell-injected mice demonstrated an increased ability to kill Renca target cells when the mice had been treated with mGITRL trimer-Fc 336B3 as compared to cells from mice treated with anti-GITR antibody or control.

Example 12 Characterization of hGITRL Trimer-Fc Proteins with and without Exogenous Peptide Linkers

A human GITRL trimer-Fc (IgG1) fusion protein was generated that replaced the stalk region (LQLETAK; SEQ ID NO:32) was an exogenous peptide linker consisting of the amino acid sequence GGGSGGG (SEQ ID NO:57). This “hGITRL trimer-Fc with linkers” construct was named 336B13 (SEQ ID NO:62 with signal sequence and SEQ ID NO:63 without signal sequence) and was used in stability studies with hGITRL trimer-Fc 336B11. The proteins were formulated in a buffer consisting of 20 mM histidine, 40 mM NaCl, 5% sucrose, and 0.01% polysorbate 20 at three different pHs (5.5, 6.0, and 6.5). The samples were stored at room temperature with analyses scheduled at time points 0, 2 weeks, and 3 months.

The samples were analyzed using Size Exclusion Chromatography-High Performance Liquid Chromatography (SEC-HPLC). This analytical assay is used to quantitate the relative purity of a protein by assessing the abundance of intact, monomeric protein versus higher molecular weight protein aggregates and/or smaller protein fragments in a sample by separating protein species by their relative size. 50 μg of protein was injected onto a Tosoh Biosciences TSK G3000SW-xl size exclusion chromatography column (7.8 mm I.D.×30 cm) and a Waters 2695 Separations Module HPLC instrument was used to run the assays.

TABLE 1 % % % % Change in Mole- Monomer Aggregate Monomer Aggregate Aggregate cule pH T = 0 T = 2 weeks (%) 336B11 5.5 96.09 3.91 96.69 3.31 −15.3 6.0 95.85 4.15 96.28 3.72 −10.4 6.5 95.78 4.22 96.35 3.65 −13.5 336B13 5.5 97.20 2.81 96.9 3.11 10.7 6.0 97.03 2.97 96.79 3.22 8.4 6.5 96.85 3.15 96.55 3.44 9.2

As shown in Table 1, although the changes in aggregation at the 2 week time point were fairly small, it appeared that hGITRL trimer-Fc 336B11 (with native stalk region) was more stable than hGITRL trimer-Fc 336B13 (with peptide linkers). 336B11 actually had less aggregates at all pHs at the 2 week time point than it did at the 0 time point. In contrast, 336B13 (with exogenous linkers) had an increased percentage of aggregates at the 2 week time point (at all pHs).

The samples were also analyzed using polyacrylamide gel electrophoresis (SDS-PAGE). This analytical assay is used to separate proteins according to their size and no other physical feature. Once the proteins are separated, the quantity of intact, monomeric protein versus higher molecular weight protein aggregates and/or smaller protein fragments in a sample can be assessed. 4 μg of protein for each sample was run on 4-20% SDS-PAGE gels under non-reduced conditions. Gel bands were detected using a Typhoon Trio imaging instrument (GE Healthcare) and quantitated by densitometry using ImageQuant TL (GE Healthcare) software. On these PAGE gels, the main, dominant band is the monomeric GITRL trimer-Fc protein, while any bands larger than the main band are aggregates and any bands smaller than the main band are fragments of the protein.

TABLE 2 % Monomer % Monomer Molecule pH T =0 T = 2 weeks 5.5 98.35 94.14 336B11 6.0 98.06 94.12 6.5 97.70 94.49 5.5 95.75 76.19 336B13 6.0 95.79 77.27 6.5 95.83 77.06

Non-reduced SDS-PAGE analysis showed little change in the percentage of the main gel band of hGITRL trimer-Fc 336B11 (with native stalk region) over the two week period at all three pHs (Table 2). In contrast, the percentage of the main gel band of hGITRL trimer-Fc 336B13 (with peptide linkers) was decreased approximately 20%, with a corresponding increase in higher and lower molecular weight bands at all three pHs (Table 2).

These results support the idea that the GITRL trimer-Fc fusion protein with the native stalk region linking the individual GITRL domains is more stable than a GITRL trimer-Fc fusion protein with exogenous peptide linkers between the individual GITRL domains and without the native stalk region.

Example 13 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein—Dose Study

Since the single chain GITRL trimer-Fc had been shown to be effective in inhibiting tumor growth, a dose range study was conducted. The murine colon tumor line CT26.WT was implanted subcutaneously (25,000 cells/mouse) in Balb/c mice and tumors were allowed to grow to an average size of approximately 115 mm³. Mice were treated with 30, 12.5, 6.25, 3, or 0.5 mg/kg of mGITRL trimer-Fc 336B3 or were untreated (n=10 per group). Mice were dosed by intraperitoneal injection twice a week for a total of 6 doses. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

FIGS. 15A-15F show the tumor volumes of individual mice within each treatment group and FIG. 15G shows the average tumor volume of each treatment group. Treatment with mGITRL trimer-Fc 336B3 strongly inhibited growth of CT26.WT tumors at each dose including at the lowest level of 0.5 mg/kg. As shown in Table 3, at Day 18 tumors had regressed to an undetectable size in at least 50% of the mice treated with 30, 12.5, 6.25, and 3 mg/kg 336B3. Even at the lowest dose of 0.5 mg/kg, total tumor regression was observed in 3 of 10 mice.

TABLE 3 Dose in mg/kg Untreated 30 12.5 6.25 3 0.5 No. of mice with 0/10 6/10 5/10 5/10 5/10 3/10 undetectable tumors at Day 18

Additional experiments were undertaken to evaluate the effect of frequency of dosing on inhibition of tumor growth by GITRL trimer. As described above, CT26.WT tumor cells were implanted subcutaneously (25,000 cells/mouse) in Balb/c mice and tumors were allowed to grow to an average size of approximately 104 mm³. Mice were treated with 2.5 mg/kg of mGITRL trimer-Fc 336B3, 12.5 mg/kg of mGITRL trimer-Fc, or were untreated (n=10 per group). Mice were treated with a single dose at 2.5 mg/kg, treated once every 2 weeks at 2.5 mg/kg, treated once every week at 2.5 mg/kg, treated twice a week at 2.5 mg/kg, or treated twice a week at 12.5 mg/kg for only 3 doses.

FIGS. 16A-16F show the tumor volumes of individual mice within each treatment group and FIG. 16G shows the average tumor volume of each treatment group. Treatment with mGITRL trimer-Fc 336B3 strongly inhibited growth of CT26.WT tumors at each dose interval. As shown in Table 4, at Day 20 tumors had regressed to an undetectable size in at least 20% of the mice treated with 2.5 mg/kg 336B3 at all dosing intervals. Even with a single dose of 336B3 at 2.5 mg/mg tumor growth was strongly inhibited and 2 mice had undetectable tumors.

TABLE 4 Dose Interval Once Q2W Q1W 2QW 2QW* Dose in mg/kg Untreated 2.5 2.5 2.5 2.5 12.5 No. of mice with 0/10 2/10 2/10 2/10 4/10 2/10 undetectable tumors at Day 20 *Mice received only 3 doses

These results demonstrate the potency of the single chain GITRL trimer as an immunotherapeutic agent. In general, these results are surprising in regard to the small amount of GITRL trimer-Fc needed to see a significant anti-tumor effect and furthermore that a significant effect is seen with limited doses. These results were unexpected, especially in light of the fact that preliminary data suggests that 336B3 has a non-linear PK and a half-life of only about 12 hours at 2 mg/kg.

Example 14 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein in Immune Cell-Depleted Mice

Experiments were conducted to evaluate which immune cell populations were involved in the inhibition of tumor growth by GITRL trimer. For in vivo depletion of specific cell populations, Balb/c mice were given an intraperitoneal injection of anti-CD4 antibody (500 ug/dose), anti-CD8 antibody (500 ug/dose), a combination of anti-CD4 and anti-CD8 antibodies (500 ug/dose each), anti-asialo GM-1 antibody (25 ul), or a control IgG2 antibody (LFT-2; 500 ug/dose) 2 days and 1 day prior to tumor cell implantation, and then additional injections were given 1 day after implantation and twice a week during the experiment. The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in the cell-depleted mice. Mice were treated on Day 7 post-implantation with 0.25 mg/mouse of mGITRL trimer-Fc 336B3 or a control antibody (n=10 per group). At Day 7, average tumor size ranged from approximately 20-50 mm³, depending on the group of cell-depleted mice. Mice were dosed by intraperitoneal injection twice a week. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

Anti-asialo GM-1 antibody was used to deplete NK cells in the treated mice, although it is known that this antibody also binds to other cells in addition to NK cells. The results from mice treated with the anti-asialo GM1 antibody are not shown as these mice were sick and all were euthanized before Day 19.

FIG. 17F shows the mean tumor volume in each group of mice and FIGS. 17A-17E show tumor volume of individual mice in each group. As seen in previous experiments, treatment with GITRL trimer-Fc 336B3 significantly inhibited tumor growth (FIG. 17B). Depletion of CD4+ cells only minimally reduced the effectiveness of GITRL-trimer-Fc 336B3 (FIG. 17C). In contrast depletion of CD8+ cells significantly affected the effectiveness of GITRL trimer-Fc 336B3 where the tumor volumes observed in the individual mice were very similar to tumor volumes seen in mice treated with the control (FIG. 17D and FIG. 17A, respectively). A higher level of tumor growth was seen in mice depleted of both CD4+ and CD8+ cells than in mice treated with control (FIG. 17E as compared to FIG. 17A).

These results demonstrate that functional CD8+ cells play a dominant role in the anti-tumor activity of the GITRL trimer-Fc 336B3. The higher level of tumor growth seen in CD4+CD8+ depleted mice indicates that the CD4+ T-cells enhance and/or are necessary for the ability of CD8+ T-cells to reduce tumor growth in the mice treated with 336B3. This result points to the importance of functional CD4+ helper T-cells for effective anti-tumor CTL activity induced by the GITRL trimer-Fc.

Example 15 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein and Anti-PD-1 Antibody

The murine adenocarcinoma cell line Renca was implanted subcutaneously (5×10⁵ cells/mouse) in Balb/c mice and on the first day of treatment (Day 7 post-implantation) the tumors were an average size of approximately 52 mm³. Mice were treated with 12.5 mg/kg of single chain mGITRL trimer-Fc 336B3, an anti-PD-1 antibody, a combination of 336B3 and anti-PD-1 antibody, or a control antibody (n=20 per group). Mice were administered 336B3 by intraperitoneal injection twice a week for only 3 doses and anti-PD-1 antibody was administered twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with electronic calipers.

As is shown in FIG. 18B, treatment with mGITRL trimer-Fc 336B3 strongly inhibited growth of the Renca tumors in a high percentage of the mice. As seen in earlier examples, treatment with 336B3 is not only able to inhibit growth of the tumors, but is able to induce regression of tumors, often to undetectable levels. Treatment with the anti-PD-1 antibody was much less successful at inhibiting tumor growth as a single agent (FIG. 18C). Treatment with the combination of GITRL trimer-Fc 336B3 and an anti-PD-1 antibody had similar results as 336B3 as a single agent (FIG. 18D).

These results support the idea that the single chain GITRL trimer is a very potent immunotherapeutic agent, even when administered as a single agent and for only a short time. In addition, the efficacy of a GITRL trimer-Fc protein may be further enhanced by combining it with other immunotherapeutic agents.

Example 16 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein and Anti-PD-L1 Antibody

The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice and on the first day of treatment (Day 10 post-implantation) the tumors were an average size of approximately 105 mm³. Mice were treated with 0.25 mg/mouse of single chain mGITRL trimer-Fc 336B3, an anti-PD-L1 antibody, a combination of 336B3 and anti-PD-L1 antibody, or a control antibody (n=10-20 per group). Mice were administered 336B3 by intraperitoneal injection twice a week for only 3 doses and anti-PD-L1 antibody was administered twice a week for 3 weeks. Tumor growth was monitored and tumor volumes were measured with electronic calipers.

As is shown in FIG. 19B, treatment with mGITRL trimer-Fc 336B3 strongly inhibited growth of the CT26.WT tumors in a high percentage of the mice. As seen in earlier examples, treatment with 336B3 is not only able to inhibit growth of the tumors, but is able to induce regression of tumors, often to undetectable levels. Treatment with the anti-PD-L1 antibody was much less successful at inhibiting tumor growth as a single agent (FIG. 19C). Treatment with the combination of GITRL trimer-Fc 336B3 and an anti-PD-L1 antibody had similar results as 336B3 as a single agent (FIG. 19D). The results of the combination treatment may be better than the GITRL trimer alone other a longer period of time.

One method of evaluating the presence and/or functionally of an anti-tumor memory cell population is to re-challenge previously treated mice with fresh tumor cells. Mice (from the studies described above) previously treated with GITRL-Fc 336B3, anti-mPD-L1 antibody, or a combination of 336B3 and anti-mPD-L1 antibody were used for a re-challenge study. Mice whose tumors had regressed completely and were undetectable at least 128 days after the first tumor injection were re-challenged with CT26.WT tumor cells (30,000 cells). The mice subjected to tumor re-challenge had received a last treatment dose 100 days prior to re-challenge. Naïve Balb/c mice (n=10) were injected with CT26.WT tumor cells (30,000 cells) as a control group. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points. Data are expressed as mean±S.E.M.

The average tumor volume of CT26.WT tumors in naive mice grew steadily up to Day 28 with an average tumor volume of approximately 1750 mm³. From the previous experiment there were only two mice with completely regressed tumors that had been previously treated with the anti-PD-L1 antibody, but these two mice demonstrated complete immunity to the tumor re-challenge. There were 13 mice with completely regressed tumors that had been previously treated with GITRL-Fc and tumors grew in only 2 of these mice after re-challenge. The other 11 mice demonstrated complete immunity to the tumor re-challenge. In addition, there were 7 mice with completely regressed tumors that had been previously treated with the combination of 336B3 and an anti-PD-L1 antibody and these mice demonstrated complete immunity to the tumor challenge. These results (as of Day 28) are shown in FIG. 19E.

The mice treated with GITRL-Fc 336B3, either as a single agent or in combination with an anti-PD-L1 antibody, appeared to be strongly protected from re-challenge with the CT26.WT tumor cells. These results suggest the existence of immunogenic memory after treatment with GITRL-Fc 336B3, either as a single agent or in combination with a checkpoint inhibitor.

Example 17 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein and Anti-PD-1 Antibody

The murine melanoma cell line B16-F10 is a poorly immunogenic tumor that originally developed in C57BL/6 mice and is thought to reflect the poor immunogenicity of metastatic tumors in humans. These cells have been shown to be unresponsive to several different types of anti-cancer therapies and therefore the B16-F10 tumor is considered to be a “high bar” model. B16-F10 cells were implanted subcutaneously (5000 cells/mouse) in C57BL/6 mice and on the first day of treatment (Day 8 post-implantation) the tumors were an average size of approximately 51 mm³. Mice were treated with 5 mg/kg of single chain mGITRL trimer-Fc 336B3, 10 mg/kg of an anti-mPD-1 antibody, a combination of 336B3 and anti-mPD-1 antibody, or a control antibody (n=10 per group). Mice were administered 336B3, anti-mPD-1 antibody, or control antibody by intraperitoneal injection twice a week. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

FIG. 20E shows the mean tumor volume in each group of mice and FIGS. 20A-20D show tumor volumes of individual mice in each group. Although treatment with the anti-PD-1 antibody had no effect on tumor growth in the B16-F10 model, treatment with GITRL trimer-Fc 336B3 was shown to have substantial efficacy in this model. When viewing the results as average tumor volume of each group, it appeared that there was only a slight increase in anti-tumor activity with the combination of 336B6 and anti-PD-1 antibody. However, when viewing the results of the individual mice, it is clear that treatment with the combination of 336B3 and anti-PD-1 inhibited tumor growth to a significant amount in 50% of the treated mice (FIG. 20D).

A follow-up study was conducted to evaluate the effective dose range of GITRL trimer-Fc 336B3 in the B16-F10 tumor model. B16-F10 cells were injected subcutaneously in C57BL/6 mice and on the first day of treatment the tumors were an average size of approximately 84 mm³. Mice were treated with 0.5 mg/kg of anti-mGITR antibody DTA-1, 30, 10, 2.5, 0.5, and 0.05 mg/kg of single chain mGITRL trimer-Fc 336B3, two different control antibodies, or saline (n=10 per group). Mice were administered 336B3, antibodies, or saline by intraperitoneal injection once a week. Tumor growth was monitored and tumor volumes were measured with electronic calipers.

The percent of mice with tumors less than 300 mm³ at study termination (Day 18) is shown in FIG. 22. Treatment with GITRL trimer-Fc 336B3 was observed to inhibit tumor growth of individual tumors at every dosage tested, including at the lowest dose of 0.05 mg/kg. In addition, in the group treated with 10 mg/kg, tumors had completely regressed in 3 mice and in the group treated with 2.5 mg/kg the tumor had completely regressed in 1 mouse.

These results are further evidence of the potent activity of the single chain GITRL trimer, even when assessed in what is considered a poorly immunogenic, “high bar” murine model.

Example 18 In Vivo Tumor Growth Inhibition by Single Chain OX40L Trimer-Fc Protein

The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice. Tumors were allowed to grow for 7 days reaching an average size of approximately 77 mm³. Mice were treated with 0.25 mg/mouse of single chain mOX40L trimer-Fc 338F2, an agonist anti-OX40 antibody, single chain mGITRL trimer-Fc, agonist anti-GITR antibody DTA-1, or a control antibody (n=10 per group). Mice were dosed twice a week for a total of 3 doses. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

As is shown in FIGS. 21D and 21E, treatment with mOX40L trimer-Fc strongly inhibited and/or prevented growth of the CT26.WT tumors. Treatment with the agonist anti-OX40 antibody also inhibited tumor growth, but to a lesser extent than the OX40L trimer-Fc molecule. A more nuanced picture of the difference in tumor growth from treatment with OX40L trimer-Fc and an OX40 antibody can be seen by looking at the results from the individual mice within each group. As shown in FIG. 21B, tumors were undetectable in 6/10 mice treated with the OX40L trimer-Fc molecule while only one mouse had an undetectable tumor in the group treated with the anti-OX40 antibody.

Similar to the results seen with GITRL trimer-Fc 336B3, these results indicate that the single chain OX40L trimer-Fc is very active as an immunotherapeutic agent and could be more potent in achieving suppression of tumor growth than an agonist OX40 antibody.

Example 19 Cytokine Production

Cytokine production after treatment with GITRL trimer-Fc, OX40L trimer-Fc protein, anti-GITR antibody, or anti-OX40 antibody was evaluated. Cells were harvested on Day 26 from the spleens of the mice described above in Example 18. The cells were cultured in the presence or the absence of the tumor specific CD8⁺ T-cell peptide AH-1. After 48 hours, cytokine levels in cell supernatants were measured using a multiplex panel for the Luminex® platform (ThermoFisher Scientific) following the manufacturer's instructions.

As shown in FIGS. 23A-23L, almost all of the cytokines measured (IL-2, IL-4, IL-5, IL-6, IL-10, IL-13, MIP-1b, FasL, GM-CSF, sCD137, granzyme B) were produced in high amounts from mice treated with agonist anti-GITR or anti-OX40 antibodies relative to the cytokine amounts from mice treated with the control antibody. This is in stark contrast to the cytokine levels produced from mice treated with the GITRL trimer-Fc or OX40L trimer-Fc proteins. Cytokine levels produced from mice treated with the GITRL-Fc or OX40L-Fc were not increased relative to control and in most cases the cytokine levels were lower than controls. In addition, preliminary analysis of plasma samples from the treated mice have shown a similar trend of increased cytokine production in mice treated with agonist anti-GITR or anti-OX40 antibodies as compared to GITRL-Fc or OX40L-Fc treated mice.

These results indicate a striking difference in impact upon immune function with the GITRL trimer-Fc and OX40L trimer-Fc proteins, compared with agonist antibodies targeting GITR and OX40. Notably, whereas the treatment with GITRL-Fc and OX40L-Fc proteins did not result in elevated levels of cytokine production by splenocytes, the broad array of cytokines which were observed to be markedly elevated following treatment with the agonist GITR and OX40 antibodies is an unanticipated immunologic result that is indicative of substantial disruption of normal immune function. Many of the cytokines observed to be upregulated following agonist antibody treatment, including IL4, IL-5, IL-6, IL-10, and IL-13 possess biological functions that would tend to decrease the capacity of the immune system to mount an appropriate Th1-type immune response that is required to develop a durable anti-tumor immune response. Importantly, elevated levels of these powerful cytokines can contribute undesirable toxicities (i.e., a cytokine storm) that may reduce the therapeutic index of the agonist antibody. As the results were similar with both the anti-GITR antibody and the anti-OX40 antibody, this finding suggests that there may be a “class-effect” common to all agonist antibodies that impact GITR or OX-40, and potentially other TNFR family members.

Example 20 In Vivo Tumor Growth Inhibition by Single Chain GITRL Trimer-Fc Protein

The murine colon tumor line CT26.WT was implanted subcutaneously (30,000 cells/mouse) in Balb/c mice. To study the effect of the GITRL trimer-Fc protein on larger, established tumor cell masses, tumors were allowed to grow until reaching an average size of approximately 300 mm³. Mice were treated with 0.25 mg/mouse of single chain mGITRL trimer-Fc 336B3, agonist anti-GITR antibody DTA-1, or a control antibody (n=17 per group). Mice were dosed twice a week for a total of 3 doses by intraperitoneal injection. Tumor growth was monitored and tumor volumes were measured with electronic calipers. As possible, mice were followed beyond 80 days to assess long-term survival.

Tumor growth for the individual mice in each group up to Day 40 is shown in FIGS. 24A-C and percent survival is shown in FIG. 24D. The growth of CT26.WT tumors was strongly inhibited and/or prevented in the mice treated with mGITRL trimer-Fc 336B3. In at least 50% of the treated mice, tumors had regressed to a size smaller than tumor size prior to treatment (FIG. 24B). The agonist anti-GITR antibody DTA-1 inhibited tumor growth in only a small number of the treated mice (FIG. 24C). Tumor growth progressed very rapidly in untreated mice with 15 mice euthanized by Day 28. The survival curve shown in FIG. 24D clearly demonstrates the effectiveness of GITRL trimer-Fc 336B3 as compared to an agonist anti-GITR antibody.

These results indicate that the single chain GITRL trimer is active as an immunotherapeutic agent even against large, established tumors and is more potent in achieving suppression of tumor growth, regression of established tumors, and increasing survival than an agonist GITR antibody.

Example 21 In Vivo Tumor Growth Inhibition in Humanized Mice by Human GITRL Trimer-Fc Protein

A humanized mouse model was used to study the efficacy of treatment with a human GITRL trimer-Fc protein on a human tumor. The humanized mice were obtained from Jackson Laboratories. These mice are created by injecting human hematopoietic stem cells (CD34+ cells) into irradiated NSG mice. After 15 weeks, the presence of mature human lymphocytes is confirmed by flow cytometry. Each mouse was injected subcutaneously with patient-derived melanoma tumor cells (OMP-M9, 75,000 cells/mouse). Tumors were allowed to grow 16 days until they had reached an average volume of approximately 60 mm³. Tumor-bearing mice were randomized into 2 groups (n=3 mice per group). Tumor-bearing mice were treated with either a control protein or hGITRL trimer-Fc OMP-336B11. Mice were dosed twice weekly at 10 mg/kg. Tumor growth was monitored and tumor volumes were measured with electronic calipers at the indicated time points.

As shown in FIG. 25, tumor growth was inhibited in the mice treated with human GITRL trimer-Fc as compared to control. These results show that the human GITRL trimer-Fc OMP-336B11 was effective at augmenting an anti-tumor immune response of human lymphocytes and contributing to inhibiting human tumor growth in vivo. Thus, these results demonstrated that humanized mouse models bearing patient-derived xenografts can be used to study the human GITRL trimer-Fc molecule in parallel with pre-clinical studies carried out with the surrogate mouse GITRL trimer-Fc protein and murine tumor models.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to person skilled in the art and are to be included within the spirit and purview of this application.

All publications, patents, patent applications, internet sites, and accession numbers/database sequences including both polynucleotide and polypeptide sequences cited herein are hereby incorporated by reference herein in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, internet site, or accession number/database sequence were specifically and individually indicated to be so incorporated by reference.

Sequences Disclosed in the application are:

Human GITRL (TNFSF18) amino acid sequence  (SEQ ID NO: 1) MTLHPSPITCEFLFSTALISPKMCLSHLENMPLSHSRTQGAQRSSWKLWLFCSIVMLLFL CSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIY GQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQV LKNNTYWGIILLANPQFIS Human GITRL signal/anchor region and extracellular domain amino acid sequence  (SEQ ID NO: 2) FCSIVMLLFLCSFSWLIFIFLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLE ILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTI DLIFNSEHQVLKNNTYWGIILLANPQFIS Human GITRL extracellular domain amino acid sequence  (SEQ ID NO: 3) LQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYN DVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGII LLANPQFIS Human single chain GITRL trimer amino acid sequence with  signal sequence underlined  (SEQ ID NO: 4) MEWGYLLEVTSLLAALLLLQRSPIVHALQLETAKEPCMAKFGPLPSKWQMASSEPPCVNK VSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYE LHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMA SSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSK IQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFG PLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDM IQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS Human single chain GITRL trimer amino acid sequence without  signal sequence  (SEQ ID NO: 5) LQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYN DVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGII LLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYG QVAPNANYNDVAPFEVRLYKNKDMIQTLINKSKIQNVGGIYELHVGDTIDLIFNSEHQVL KNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEIL QNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDL IFNSEHQVLKNNTYWGIILLANPQFIS 336B11 Human single chain GITRL trimer-Fc (IgG1) amino acid  sequence with signal sequence underlined  (SEQ ID NO: 6) MEWGYLLEVTSLLAALLLLQRSPIVHALQLETAKEPCMAKFGPLPSKWQMASSEPPCVNK VSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYE LHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMA SSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSK IQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFG PLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDM IQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 336B11 Human single chain GITRL trimer-Fc (IgG1) amino acid  sequence without signal sequence (SEQ ID NO: 7) LQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYN DVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGII LLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYG QVAPNANYNDVAPFEVRLYKNKDMIQTLINKSKIQNVGGIYELHVGDTIDLIFNSEHQVL KNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEIL QNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDL IFNSEHQVLKNNTYWGIILLANPQFISDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKITPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK 336B14 Human single chain GITRL trimer-Fc (IgG2) amino acid  sequence with signal sequence underlined (SEQ ID NO: 8) MEWGYLLEVTSLLAALLLLQRSPIVHALQLETAKEPCMAKFGPLPSKWQMASSEPPCVNK VSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYE LHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMA SSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSK IQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFG PLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDM IQTLINKSKIQNVGGIYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISVERKSC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 336B14 Human single chain GITRL trimer-Fc (IgG2) amino acid  sequence without signal sequence (SEQ ID NO: 9) LQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYN DVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGII LLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYG QVAPNANYNDVAPFEVRLYKNKDMIQTLINKSKIQNVGGIYELHVGDTIDLIFNSEHQVL KNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEIL QNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDL IFNSEHQVLKNNTYWGIILLANPQFISVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKITPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK Human IgG1 Fc region  (SEQ ID NO: 10) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region  (SEQ ID NO: 11) KSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region  (SEQ ID NO: 12) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVICVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region  (SEQ ID NO: 13) CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region  (SEQ ID NO: 14) VERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRIPEVTCVVVDVSHEDPEVQFNWY VDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISK TKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPML DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Heavy chain constant region  (SEQ ID NO: 15) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDE LTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Heavy chain constant region  (SEQ ID NO: 16) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR VVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Human IgG3 Heavy chain constant region  (SEQ ID NO: 17) ASTKGPSVFPLAPCSRSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTQTYTCNVNHKPSNTKVDKRVELKTPLGDTTHTCPRCPEPKSC DTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPAPELLGGPSVFLFPPKPKDT LMISRIPEVICVVVDVSHEDPEVQFKWYVDGVEVHNAKTKPREEQYNSTFRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESSGQPENNYNTTPPMLDSDGSFFLYSKLTVDKSRWQQGNIFSCSVMHE ALHNRFTQKSLSLSPGK Human IgG4 Heavy chain constant region  (SEQ ID NO: 18) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS GLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSV FLFPPKPKDTLMISRIPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTY RVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEG NVFSCSVMHEALHNHYTQKSLSLSLGK Human GITRL nucleotide sequence (SEQ ID NO: 19) ATGACATTGCATCCTTCACCCATCACTTGTGAATTTTTGTTTTCCACAGCTCTCATTTCT CCAAAAATGTGTTTGAGCCACTTGGAAAATATGCCTTTAAGCCATTCAAGAACTCAAGGA GCTCAGAGATCATCCTGGAAGCTGTGGCTCTTTTGCTCAATAGTTATGTTGCTATTTCTT TGCTCCTTCAGTTGGCTAATCTTTATTTTTCTCCAATTAGAGACTGCTAAGGAGCCCTGT ATGGCTAAGTTTGGACCATTACCCTCAAAATGGCAAATGGCATCTTCTGAACCTCCTTGC GTGAATAAGGTGTCTGACTGGAAGCTGGAGATACTTCAGAATGGCTTATATTTAATTTAT GGCCAAGTGGCTCCCAATGCAAACTACAATGATGTAGCTCCTTTTGAGGTGCGGCTGTAT AAAAACAAAGACATGATACAAACTCTAACAAACAAATCTAAAATCCAAAATGTAGGAGGG ACTTATGAATTGCATGTTGGGGACACCATAGACTTGATATTCAACTCTGAGCATCAGGTT CTAAAAAATAATACATACTGGGGTATCATTTTACTAGCAAATCCCCAATTCATCTCCTAG Human GITRL signal/anchor region and extracellular domain  nucleotide sequence  (SEQ ID NO: 20) TTTTGCTCAATAGTTATGTTGCTATTTCTTTGCTCCTTCAGTTGGCTAATCTTTATTTTT CTCCAATTAGAGACTGCTAAGGAGCCCTGTATGGCTAAGTTTGGACCATTACCCTCAAAA TGGCAAATGGCATCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAG ATACTTCAGAATGGCTTATATTTAATTTATGGCCAAGTGGCTCCCAATGCAAACTACAAT GATGTAGCTCCTTTTGAGGTGCGGCTGTATAAAAACAAAGACATGATACAAACTCTAACA AACAAATCTAAAATCCAAAATGTAGGAGGGACTTATGAATTGCATGTTGGGGACACCATA GACTTGATATTCAACTCTGAGCATCAGGTTCTAAAAAATAATACATACTGGGGTATCATT TTACTAGCAAATCCCCAATTCATCTCCTAG Human GITRL extracellular domain nucleotide sequence  (SEQ ID NO: 21) CTCCAATTAGAGACTGCTAAGGAGCCCTGTATGGCTAAGTTTGGACCATTACCCTCAAAA TGGCAAATGGCATCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAG ATACTTCAGAATGGCTTATATTTAATTTATGGCCAAGTGGCTCCCAATGCAAACTACAAT GATGTAGCTCCTTTTGAGGTGCGGCTGTATAAAAACAAAGACATGATACAAACTCTAACA AACAAATCTAAAATCCAAAATGTAGGAGGGACTTATGAATTGCATGTTGGGGACACCATA GACTTGATATTCAACTCTGAGCATCAGGTTCTAAAAAATAATACATACTGGGGTATCATT TTACTAGCAAATCCCCAATTCATCTCCTAG Human single chain GITRL trimer without signal sequence  nucleotide sequence  (SEQ ID NO: 22) CTGCAACTGGAAACCGCTAAGGAGCCCTGTATGGCTAAGTTCGGCCCACTGCCTTCCAAA TGGCAGATGGCATCTAGTGAGCCACCCTGTGTTAATAAAGTTAGCGATTGGAAACTGGAG ATCCTGCAAAACGGGCTCTACCTGATTTACGGACAAGTTGCTCCTAATGCTAACTACAAT GATGTGGCTCCTTTTGAAGTTAGGCTGTATAAAAACAAAGACATGATCCAAACTCTCACT AACAAAAGCAAAATCCAAAATGTCGGTGGGACTTATGAGCTCCATGTTGGGGACACCATC GACCTGATTTTCAACTCTGAGCATCAGGTTCTCAAAAATAATACATACTGGGGAATCATT CTCCTCGCGAATCCACAATTCATCTCTCTCCAACTGGAAACCGCTAAAGAACCTTGCATG GCCAAATTTGGACCTCTCCCAAGCAAATGGCAAATGGCTTCTTCTGAACCTCCTTGCGTG AATAAGGTGTCTGACTGGAAGCTGGAGATTCTGCAGAATGGCCTCTATCTGATTTATGGG CAAGTTGCACCTAACGCTAATTATAACGACGTCGCACCATTCGAAGTTCGCCTCTACAAA AATAAGGACATGATTCAAACACTGACTAATAAATCCAAAATTCAAAACGTTGGGGGCACA TACGAACTGCACGTCGGCGATACTATTGATCTCATCTTTAATTCCGAACACCAGGTCCTC AAAAACAATACCTATTGGGGGATCATCCTCCTGGCTAACCCACAATTTATATCTCTCCAA CTCGAAACAGCCAAGGAACCATGTATGGCAAAGTTTGGTCCCCTCCCATCCAAGTGGCAA ATGGCCAGTTCTGAACCCCCATGCGTTAATAAGGTTTCCGACTGGAAACTGGAGATCCTG CAAAATGGTCTGTACCTCATCTATGGTCAAGTTGCACCAAACGCCAATTACAATGATGTT GCACCATTTGAAGTTCGCCTGTACAAAAACAAAGATATGATCCAAACCCTCACTAACAAA TCTAAAATCCAAAATGTTGGTGGTACTTACGAACTGCATGTGGGTGACACCATCGACCTC ATCTTCAATTCCGAGCATCAGGTGCTCAAAAACAATACATATTGGGGCATAATTCTGCTC GCAAATCCACAATTCATCTCT Human single chain GITRL trimer with signal sequence  nucleotide sequence  (SEQ ID NO: 23) ATGGAGTGGGGTTATCTGCTCGAAGTGACCTCCCTGCTGGCCGCCCTGCTCCTGCTGCAA CGCTCTCCTATCGTGCACGCCCTGCAACTGGAAACCGCTAAGGAGCCCTGTATGGCTAAG TTCGGCCCACTGCCTTCCAAATGGCAGATGGCATCTAGTGAGCCACCCTGTGTTAATAAA GTTAGCGATTGGAAACTGGAGATCCTGCAAAACGGGCTCTACCTGATTTACGGACAAGTT GCTCCTAATGCTAACTACAATGATGTGGCTCCTTTTGAAGTTAGGCTGTATAAAAACAAA GACATGATCCAAACTCTCACTAACAAAAGCAAAATCCAAAATGTCGGTGGGACTTATGAG CTCCATGTTGGGGACACCATCGACCTGATTTTCAACTCTGAGCATCAGGTTCTCAAAAAT AATACATACTGGGGAATCATTCTCCTCGCGAATCCACAATTCATCTCTCTCCAACTGGAA ACCGCTAAAGAACCTTGCATGGCCAAATTTGGACCTCTCCCAAGCAAATGGCAAATGGCT TCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAGATTCTGCAGAAT GGCCTCTATCTGATTTATGGGCAAGTTGCACCTAACGCTAATTATAACGACGTCGCACCA TTCGAAGTTCGCCTCTACAAAAATAAGGACATGATTCAAACACTGACTAATAAATCCAAA ATTCAAAACGTTGGGGGCACATACGAACTGCACGTCGGCGATACTATTGATCTCATCTTT AATTCCGAACACCAGGTCCTCAAAAACAATACCTATTGGGGGATCATCCTCCTGGCTAAC CCACAATTTATATCTCTCCAACTCGAAACAGCCAAGGAACCATGTATGGCAAAGTTTGGT CCCCTCCCATCCAAGTGGCAAATGGCCAGTTCTGAACCCCCATGCGTTAATAAGGTTTCC GACTGGAAACTGGAGATCCTGCAAAATGGTCTGTACCTCATCTATGGTCAAGTTGCACCA AACGCCAATTACAATGATGTTGCACCATTTGAAGTTCGCCTGTACAAAAACAAAGATATG ATCCAAACCCTCACTAACAAATCTAAAATCCAAAATGTTGGTGGTACTTACGAACTGCAT GTGGGTGACACCATCGACCTCATCTTCAATTCCGAGCATCAGGTGCTCAAAAACAATACA TATTGGGGCATAATTCTGCTCGCAAATCCACAATTCATCTCT 336B11 Human single chain GITRL trimer-Fc (IgG1) nucleotide  sequence  (SEQ ID NO: 24) ATGGAGTGGGGTTATCTGCTCGAAGTGACCTCCCTGCTGGCCGCCCTGCTCCTGCTGCAA CGCTCTCCTATCGTGCACGCCCTGCAACTGGAAACCGCTAAGGAGCCCTGTATGGCTAAG TTCGGCCCACTGCCTTCCAAATGGCAGATGGCATCTAGTGAGCCACCCTGTGTTAATAAA GTTAGCGATTGGAAACTGGAGATCCTGCAAAACGGGCTCTACCTGATTTACGGACAAGTT GCTCCTAATGCTAACTACAATGATGTGGCTCCTTTTGAAGTTAGGCTGTATAAAAACAAA GACATGATCCAAACTCTCACTAACAAAAGCAAAATCCAAAATGTCGGTGGGACTTATGAG CTCCATGTTGGGGACACCATCGACCTGATTTTCAACTCTGAGCATCAGGTTCTCAAAAAT AATACATACTGGGGAATCATTCTCCTCGCGAATCCACAATTCATCTCTCTCCAACTGGAA ACCGCTAAAGAACCTTGCATGGCCAAATTTGGACCTCTCCCAAGCAAATGGCAAATGGCT TCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAGATTCTGCAGAAT GGCCTCTATCTGATTTATGGGCAAGTTGCACCTAACGCTAATTATAACGACGTCGCACCA TTCGAAGTTCGCCTCTACAAAAATAAGGACATGATTCAAACACTGACTAATAAATCCAAA ATTCAAAACGTTGGGGGCACATACGAACTGCACGTCGGCGATACTATTGATCTCATCTTT AATTCCGAACACCAGGTCCTCAAAAACAATACCTATTGGGGGATCATCCTCCTGGCTAAC CCACAATTTATATCTCTCCAACTCGAAACAGCCAAGGAACCATGTATGGCAAAGTTTGGT CCCCTCCCATCCAAGTGGCAAATGGCCAGTTCTGAACCCCCATGCGTTAATAAGGTTTCC GACTGGAAACTGGAGATCCTGCAAAATGGTCTGTACCTCATCTATGGTCAAGTTGCACCA AACGCCAATTACAATGATGTTGCACCATTTGAAGTTCGCCTGTACAAAAACAAAGATATG ATCCAAACCCTCACTAACAAATCTAAAATCCAAAATGTTGGTGGTACTTACGAACTGCAT GTGGGTGACACCATCGACCTCATCTTCAATTCCGAGCATCAGGTGCTCAAAAACAATACA TATTGGGGCATAATTCTGCTCGCAAATCCACAATTCATCTCTGACAAGACCCACACCTGC CCTCCCTGCCCTGCCCCTGAGCTGCTGGGCGGACCTTCCGTGTTCCTGTTCCCTCCTAAG CCTAAGGACACCCTGATGATCTCCCGGACCCCTGAAGTGACATGCGTGGTGGTGGACGTG TCCCACGAGGACCCTGAGGTGAAGTTCAACTGGTATGTGGACGGCGTGGAGGTGCACAAC GCTAAGACCAAGCCTAGGGAGGAGCAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTG ACCGTGCTGCACCAGGACTGGCTGAACGGCAAAGAATACAAGTGCAAGGTCTCCAACAAG GCCCTGCCCGCTCCCATCGAGAAAACCATCAGCAAGGCAAAGGGCCAGCCTCGCGAGCCT CAGGTGTACACCCTGCCACCCAGCCGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACC TGTCTGGTGAAGGGCTTTTACCCTTCCGATATTGCCGTGGAGTGGGAGTCTAACGGCCAG CCCGAGAACAACTACAAGACCACCCCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTG TACTCCAAGCTGACCGTGGACAAGTCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCC GTGATGCACGAGGCCCTGCACAACCACTACACCCAGAAGAGCCTGTCTCTGTCTCCTGGC AAGTGA 336B14 Human single chain GITRL trimer-Fc (IgG2) nucleotide  sequence  (SEQ ID NO: 25) ATGGAGTGGGGTTATCTGCTCGAAGTGACCTCCCTGCTGGCCGCCCTGCTCCTGCTGCAA CGCTCTCCTATCGTGCACGCCCTGCAACTGGAAACCGCTAAGGAGCCCTGTATGGCTAAG TTCGGCCCACTGCCTTCCAAATGGCAGATGGCATCTAGTGAGCCACCCTGTGTTAATAAA GTTAGCGATTGGAAACTGGAGATCCTGCAAAACGGGCTCTACCTGATTTACGGACAAGTT GCTCCTAATGCTAACTACAATGATGTGGCTCCTTTTGAAGTTAGGCTGTATAAAAACAAA GACATGATCCAAACTCTCACTAACAAAAGCAAAATCCAAAATGTCGGTGGGACTTATGAG CTCCATGTTGGGGACACCATCGACCTGATTTTCAACTCTGAGCATCAGGTTCTCAAAAAT AATACATACTGGGGAATCATTCTCCTCGCGAATCCACAATTCATCTCTCTCCAACTGGAA ACCGCTAAAGAACCTTGCATGGCCAAATTTGGACCTCTCCCAAGCAAATGGCAAATGGCT TCTTCTGAACCTCCTTGCGTGAATAAGGTGTCTGACTGGAAGCTGGAGATTCTGCAGAAT GGCCTCTATCTGATTTATGGGCAAGTTGCACCTAACGCTAATTATAACGACGTCGCACCA TTCGAAGTTCGCCTCTACAAAAATAAGGACATGATTCAAACACTGACTAATAAATCCAAA ATTCAAAACGTTGGGGGCACATACGAACTGCACGTCGGCGATACTATTGATCTCATCTTT AATTCCGAACACCAGGTCCTCAAAAACAATACCTATTGGGGGATCATCCTCCTGGCTAAC CCACAATTTATATCTCTCCAACTCGAAACAGCCAAGGAACCATGTATGGCAAAGTTTGGT CCCCTCCCATCCAAGTGGCAAATGGCCAGTTCTGAACCCCCATGCGTTAATAAGGTTTCC GACTGGAAACTGGAGATCCTGCAAAATGGTCTGTACCTCATCTATGGTCAAGTTGCACCA AACGCCAATTACAATGATGTTGCACCATTTGAAGTTCGCCTGTACAAAAACAAAGATATG ATCCAAACCCTCACTAACAAATCTAAAATCCAAAATGTTGGTGGTACTTACGAACTGCAT GTGGGTGACACCATCGACCTCATCTTCAATTCCGAGCATCAGGTGCTCAAAAACAATACA TATTGGGGCATAATTCTGCTCGCAAATCCACAATTCATCTCTGTTGAGCGCAAATCTTGT GTCGAGTGCCCACCTTGCCCAGCACCACCTGTGGCAGGACCTTCAGTCTTCCTCTTCCCC CCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCCGAGGTCCAGTTTAATTGGTATGTCGACGGCGTGGAGGTG CATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACATTCAGGGTGGTCAGC GTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTGTCC AACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGCCCAGA GAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC CTGACCTGCCTGGTGAAGGGATTTTATCCTTCCGACATCGCCGTGGAGTGGGAGAGCAAT GGGCAGCCTGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTC TTCCTGTATTCCAAACTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCA TGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCC CCTGGAAAGTGA Human IgG2 Fc region (13A Version)  (SEQ ID NO: 26) CVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLKSDGS FFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region (13B Version)  (SEQ ID NO: 27) CVECPPCPAPPVAGPSVFLFPPKPEODTLMISRTPEVTCWVDVSHEDPEVQFNWYVDGVE VHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQP REPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGS FFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region (13A Version)  (SEQ ID NO: 28) TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREKMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region (13A Version)  (SEQ ID NO: 29) TKVDFCTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPMLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region (13B Version)  (SEQ ID NO: 30) TKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG2 Fc region (13B Version)  (SEQ ID No: 31) TKVDKTVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human GITRL Stalk Region  (SEQ ID NO: 32) LQLETAK Human GITRL TNF Homology Domain  (SEQ ID NO: 33) KWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTL TNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILL Linker  (SEQ ID NO: 34) ESGGGGVT Linker  (SEQ ID NO: 35) LESGGGGVT Linker  (SEQ ID NO: 36) GRAQVT Linker  (SEQ ID NO: 37) WRAQVT Linker  (SEQ ID NO: 38) ARGRAQVT FLAG Tag  (SEQ ID NO: 39) DYKDDDDK Human OX40L (TNFSF4) amino acid sequence  (SEQ ID NO: 40) MERVQPLEENVGNAARPRFERNKLLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQ SIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQ KDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEF CVL Human OX40L transmembrane and extracellular domain amino acid sequence  (SEQ ID NO: 4l) LLLVASVIQGLGLLLCFTYICLHFSALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED EIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL Human OX40L extracellular domain amino acid sequence  (SEQ ID NO: 42) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEWISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVL Human single chain OX40L trimer amino acid sequence with  signal sequence underlined  (SEQ ID NO: 43) MEWGYLLEVTSLLAALLLLQRSPIVHAQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED EIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYK KEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLK EWRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYP RIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISL HYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNP GEFCVL Human single chain OX40L trimer amino acid sequence without  signal sequence  (SEQ ID NO: 44) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINC DGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDN TSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDE IMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLT YKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 338F3 Human single chain OX40L trimer-Fc (IgG1) amino acid  sequence with signal sequence underlined  (SEQ ID NO: 45) MEWGYLLEVTSLLAALLLLQRSPIVHAQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED EIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYK KEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLK KVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYP RIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISL HYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNP GEFCVLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVK FNWYVTCVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 338F3 Human single chain OX40L trimer-Fc (IgG1) amino acid  sequence without signal sequence  (SEQ ID NO: 46) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINC DGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDN TSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDE IMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLT YKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLDKTHTCPPCPAPELLGGPSVF LFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKN QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKSLSLSPGK Human single chain OX40L trimer-Fc (IgG2) amino acid  sequence with signal sequence underlined (SEQ ID NO: 47) MEWGYLLEVTSLLAALLLLQRSPIVHAQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED EIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYK KEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLK EWRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYP RIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISL HYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNP GEFCVLVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPI EKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYK TTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human single chain OX40L trimer-Fc (IgG2) amino acid   sequence without signal sequence  (SEQ ID NO: 48) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINC DGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDN TSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDE IMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLT YKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLVERKSCVECPPCPAPPVAGPS VFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNST FRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMT KNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ GNVFSCSVMHEALHNHYTQKSLSLSPGK Human OX40L nucleotide sequence  (SEQ ID NO: 49) ATGGAAAGGGTCCAACCCCTGGAAGAGAATGTGGGAAATGCAGCCAGGCCAAGATTCGAG AGGAACAAGCTATTGCTGGTGGCCTCTGTAATTCAGGGACTGGGGCTGCTCCTGTGCTTC ACCTACATCTGCCTGCACTTCTCTGCTCTTCAGGTATCACATCGGTATCCTCGAATTCAA AGTATCAAAGTACAATTTACCGAATATAAGAAGGAGAAAGGTTTCATCCTCACTTCCCAA AAGGAGGATGAAATCATGAAGGTGCAGAACAACTCAGTCATCATCAACTGTGATGGGTTT TATCTCATCTCCCTGAAGGGCTACTTCTCCCAGGAAGTCAACATTAGCCTTCATTACCAG AAGGATGAGGAGCCCCTCTTCCAACTGAAGAAGGTCAGGTCTGTCAACTCCTTGATGGTG GCCTCTCTGACTTACAAAGACAAAGTCTACTTGAATGTGACCACTGACAATACCTCCCTG GATGACTTCCATGTGAATGGCGGAGAACTGATTCTTATCCATCAAAATCCTGGTGAATTC TGTGTCCTTTGA Human OX40L extracellular domain nucleotide sequence  (SEQ ID NO: 50) CAGGTATCACATCGGTATCCTCGAATTCAAAGTATCAAAGTACAATTTACCGAATATAAG AAGGAGAAAGGTTTCATCCTCACTTCCCAAAAGGAGGATGAAATCATGAAGGTGCAGAAC AACTCAGTCATCATCAACTGTGATGGGTTTTATCTCATCTCCCTGAAGGGCTACTTCTCC CAGGAAGTCAACATTAGCCTTCATTACCAGAAGGATGAGGAGCCCCTCTTCCAACTGAAG AAGGTCAGGTCTGTCAACTCCTTGATGGTGGCCTCTCTGACTTACAAAGACAAAGTCTAC TTGAATGTGACCACTGACAATACCTCCCTGGATGACTTCCATGTGAATGGCGGAGAACTG ATTCTTATCCATCAAAATCCTGGTGAATTCTGTGTCCTTTGA Human single chain OX40L trimer with signal sequence  nucleotide sequence (SEQ ID NO: 51) ATGGAGTGGGGTTATCTGCTCGAAGTGACCTCCCTGCTGGCCGCCCTGCTCCTGCTGCAA CGCTCTCCTATCGTGCACGCCCAGGTCTCTCATAGATACCCACGCATACAATCAATCAAA GTCCAATTTACAGAATATAAGAAGGAAAAAGGATTCATTCTCACATCTCAGAAGGAGGAC GAAATCATGAAAGTGCAAAATAACAGCGTGATAATTAATTGCGACGGTTTTTACCTGATC TCCCTGAAGGGGTATTTCTCCCAGGAGGTCAATATTTCTCTCCACTACCAAAAGGACGAG GAACCTCTCTTCCAGCTCAAGAAAGTTAGAAGCGTCAATTCCCTGATGGTGGCCTCCCTG ACTTATAAGGATAAAGTGTATCTCAATGTTACCACAGATAACACTTCTCTGGATGATTTC CATGTCAATGGTGGAGAGCTCATCCTCATTCACCAGAACCCTGGGGAGTTCTGCGTACTG CAAGTCTCACACCGGTACCCGCGCATCCAAAGCATAAAAGTTCAATTCACCGAGTATAAA AAAGAGAAGGGTTTCATACTCACATCACAAAAGGAAGATGAAATTATGAAGGTTCAAAAC AACTCTGTTATCATTAACTGCGATGGGTTCTATCTGATTTCACTGAAAGGTTACTTCAGC CAAGAGGTGAACATATCTCTGCATTATCAGAAAGATGAAGAGCCCCTGTTCCAACTGAAG AAGGTCCGCTCAGTCAACTCACTGATGGTTGCATCCCTCACATATAAAGATAAGGTCTAT CTGAATGTGACAACTGACAATACCTCACTGGATGACTTTCATGTTAACGGAGGCGAACTG ATTCTCATACATCAGAATCCAGGAGAGTTCTGTGTCCTCCAAGTTTCCCATCGCTATCCT CGGATTCAATCTATCAAGGTTCAGTTTACTGAGTACAAAAAAGAAAAGGGATTTATTCTG ACCTCTCAAAAAGAGGATGAGATAATGAAGGTCCAGAATAATTCCGTCATTATAAACTGT GACGGCTTCTATCTCATATCCCTCAAGGGGTACTTTTCACAAGAAGTTAATATATCACTC CATTACCAAAAAGATGAAGAGCCACTCTTTCAACTGAAAAAAGTCAGATCCGTCAACTCT CTCATGGTCGCTTCTCTCACCTACAAAGACAAAGTTTACCTGAACGTTACTACAGACAAC ACATCCCTGGACGACTTCCACGTGAATGGCGGGGAACTGATACTGATCCACCAAAATCCC GGCGAATTTTGTGTGCTC Human single chain OX40L trimer without signal sequence  nucleotide sequence  (SEQ ID NO: 52) CAGGTCTCTCATAGATACCCACGCATACAATCAATCAAAGTCCAATTTACAGAATATAAG AAGGAAAAAGGATTCATTCTCACATCTCAGAAGGAGGACGAAATCATGAAAGTGCAAAAT AACAGCGTGATAATTAATTGCGACGGTTTTTACCTGATCTCCCTGAAGGGGTATTTCTCC CAGGAGGTCAATATTTCTCTCCACTACCAAAAGGACGAGGAACCTCTCTTCCAGCTCAAG AAAGTTAGAAGCGTCAATTCCCTGATGGTGGCCTCCCTGACTTATAAGGATAAAGTGTAT CTCAATGTTACCACAGATAACACTTCTCTGGATGATTTCCATGTCAATGGTGGAGAGCTC ATCCTCATTCACCAGAACCCTGGGGAGTTCTGCGTACTGCAAGTCTCACACCGGTACCCG CGCATCCAAAGCATAAAAGTTCAATTCACCGAGTATAAAAAAGAGAAGGGTTTCATACTC ACATCACAAAAGGAAGATGAAATTATGAAGGTTCAAAACAACTCTGTTATCATTAACTGC GATGGGTTCTATCTGATTTCACTGAAAGGTTACTTCAGCCAAGAGGTGAACATATCTCTG CATTATCAGAAAGATGAAGAGCCCCTGTTCCAACTGAAGAAGGTCCGCTCAGTCAACTCA CTGATGGTTGCATCCCTCACATATAAAGATAAGGTCTATCTGAATGTGACAACTGACAAT ACCTCACTGGATGACTTTCATGTTAACGGAGGCGAACTGATTCTCATACATCAGAATCCA GGAGAGTTCTGTGTCCTCCAAGTTTCCCATCGCTATCCTCGGATTCAATCTATCAAGGTT CAGTTTACTGAGTACAAAAAAGAAAAGGGATTTATTCTGACCTCTCAAAAAGAGGATGAG ATAATGAAGGTCCAGAATAATTCCGTCATTATAAACTGTGACGGCTTCTATCTCATATCC CTCAAGGGGTACTTTTCACAAGAAGTTAATATATCACTCCATTACCAAAAAGATGAAGAG CCACTCTTTCAACTGAAAAAAGTCAGATCCGTCAACTCTCTCATGGTCGCTTCTCTCACC TACAAAGACAAAGTTTACCTGAACGTTACTACAGACAACACATCCCTGGACGACTTCCAC GTGAATGGCGGGGAACTGATACTGATCCACCAAAATCCCGGCGAATTTTGTGTGCTC Human single chain OX40L-Fc (IgG1) trimer nucleotide   sequence (SEQ ID NO: 53) ATGGAGTGGGGTTATCTGCTCGAAGTGACCTCCCTGCTGGCCGCCCTGCTCCTGCTGCAA CGCTCTCCTATCGTGCACGCCCAGGTCTCTCATAGATACCCACGCATACAATCAATCAAA GTCCAATTTACAGAATATAAGAAGGAAAAAGGATTCATTCTCACATCTCAGAAGGAGGAC GAAATCATGAAAGTGCAAAATAACAGCGTGATAATTAATTGCGACGGTTTTTACCTGATC TCCCTGAAGGGGTATTTCTCCCAGGAGGTCAATATTTCTCTCCACTACCAAAAGGACGAG GAACCTCTCTTCCAGCTCAAGAAAGTTAGAAGCGTCAATTCCCTGATGGTGGCCTCCCTG ACTTATAAGGATAAAGTGTATCTCAATGTTACCACAGATAACACTTCTCTGGATGATTTC CATGTCAATGGTGGAGAGCTCATCCTCATTCACCAGAACCCTGGGGAGTTCTGCGTACTG CAAGTCTCACACCGGTACCCGCGCATCCAAAGCATAAAAGTTCAATTCACCGAGTATAAA AAAGAGAAGGGTTTCATACTCACATCACAAAAGGAAGATGAAATTATGAAGGTTCAAAAC AACTCTGTTATCATTAACTGCGATGGGTTCTATCTGATTTCACTGAAAGGTTACTTCAGC CAAGAGGTGAACATATCTCTGCATTATCAGAAAGATGAAGAGCCCCTGTTCCAACTGAAG AAGGTCCGCTCAGTCAACTCACTGATGGTTGCATCCCTCACATATAAAGATAAGGTCTAT CTGAATGTGACAACTGACAATACCTCACTGGATGACTTTCATGTTAACGGAGGCGAACTG ATTCTCATACATCAGAATCCAGGAGAGTTCTGTGTCCTCCAAGTTTCCCATCGCTATCCT CGGATTCAATCTATCAAGGTTCAGTTTACTGAGTACAAAAAAGAAAAGGGATTTATTCTG ACCTCTCAAAAAGAGGATGAGATAATGAAGGTCCAGAATAATTCCGTCATTATAAACTGT GACGGCTTCTATCTCATATCCCTCAAGGGGTACTTTTCACAAGAAGTTAATATATCACTC CATTACCAAAAAGATGAAGAGCCACTCTTTCAACTGAAAAAAGTCAGATCCGTCAACTCT CTCATGGTCGCTTCTCTCACCTACAAAGACAAAGTTTACCTGAACGTTACTACAGACAAC ACATCCCTGGACGACTTCCACGTGAATGGCGGGGAACTGATACTGATCCACCAAAATCCC GGCGAATTTTGTGTGCTCGACAAGACCCACACCTGCCCTCCCTGCCCTGCCCCTGAGCTG CTGGGCGGACCTTCCGTGTTCCTGTTCCCTCCTAAGCCTAAGGACACCCTGATGATCTCC CGGACCCCTGAAGTGACATGCGTGGTGGTGGACGTGTCCCACGAGGACCCTGAGGTGAAG TTCAACTGGTATGTGGACGGCGTGGAGGTGCACAACGCTAAGACCAAGCCTAGGGAGGAG CAGTACAACTCCACCTACCGGGTGGTGTCTGTGCTGACCGTGCTGCACCAGGACTGGCTG AACGGCAAAGAATACAAGTGCAAGGTCTCCAACAAGGCCCTGCCCGCTCCCATCGAGAAA ACCATCAGCAAGGCAAAGGGCCAGCCTCGCGAGCCTCAGGTGTACACCCTGCCACCCAGC CGGGAGGAGATGACCAAGAACCAGGTGTCCCTGACCTGTCTGGTGAAGGGCTTTTACCCT TCCGATATTGCCGTGGAGTGGGAGTCTAACGGCCAGCCCGAGAACAACTACAAGACCACC CCTCCTGTGCTGGACTCCGACGGCTCCTTCTTCCTGTACTCCAAGCTGACCGTGGACAAG TCCCGGTGGCAGCAGGGCAACGTGTTCTCCTGCTCCGTGATGCACGAGGCCCTGCACAAC CACTACACCCAGAAGAGCCTGTCTCTGTCTCCTGGCAAGTGA AH-1 Peptide  (SEQ ID NO: 54) SPSYVYHQF OX40L Stalk region  (SEQ ID NO: 55) QVSHRYP OX40L TNF homology domain  (SEQ ID NO: 56) EIMEWQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPG Linker  (SEQ ID NO: 57) GGGSGGG Human IgG1 Fc region (13A Version)  (SEQ ID NO: 58) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region (13B Version)  (SEQ ID NO: 59) DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region (13A Version)  (SEQ ID NO: 60) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDKLTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTP PVLKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human IgG1 Fc region (13B Version)  (SEQ ID NO: 61) EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVEGFYPSDIAVEWESNGQPENNYKTTP PVLDSDGSFFLYSELTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 336B13 Human single chain GITRL trimer-Fc (IgG1) with linkers with signal sequence underlined (SEQ ID NO: 62) MEWGYLLEVTSLLAALLLLQRSPIVHALQLETAKEPCMAKFGPLPSKWQMASSEPPCVNK VSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYE LHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISGGGSGGGEPCMAKFGPLPSKWQMA SSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSK IQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISGGGSGGGEPCMAKFG PLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDM IQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 336B13 Human single chain GITRL trimer-Fc (IgG1) with linkers without signal sequence  (SEQ ID NO: 63) LQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYN DVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGII LLANPQFISGGGSGGGEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYG QVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVL KNNTYWGIILLANPQFISGGGSGGGEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEIL QNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDL IFNSEHQVLKNNTYWGIILLANPQFISDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK Human GITRL extracellular domain without stalk region amino  acid sequence  (SEQ ID NO: 64) EPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEV RLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQF IS Human single chain GITRL trimer (2 stalk regions) amino acid sequence with signal sequence underlined (SEQ ID NO: 65) MEWGYLLEVTSLLAALLLLQRSPIVHAEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLE ILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTI DLIFNSEHQVLKIWTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCV NKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGT YELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFISLQLETAKEPCMAKFGPLPSKWQ MASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNK SKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQFIS Human single chain GITRL trimer (2 stalk regions) amino acid sequence without signal sequence  (SEQ ID NO: 66) EPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNANYNDVAPFEV RLYKNKDMIQTLTNKSKIQKVGGTYELHVGDTIDLIFNSEHQVLKNNTYWGIILLANPQF ISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLIYGQVAPNAN YNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQVLKNNTYWG IILLANPQFISLQLETAKEPCMAKFGPLPSKWQMASSEPPCVNKVSDWKLEILQNGLYLI YGQVAPNANYNDVAPFEVRLYKNKDMIQTLTNKSKIQNVGGTYELHVGDTIDLIFNSEHQ VLKNNTYWGIILLANPQFIS Human OX40L extracellular domain without stalk region amino  acid sequence  (SEQ ID NO: 67) RIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISL HYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNP GEFCVL Human single chain OX40L trimer (2 stalk regions) amino acid sequence with signal sequence underlined (SEQ ID NO: 68) MEWGYLLEVTSLLAALLLLQRSPIVHARIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQN NSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVY LNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFIL TSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNS LMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLQVSHRYPRIQSIKV QFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEE PLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL Human single chain OX40L trimer (2 stalk regions) amino acid  sequence without signal sequence  (SEQ ID NO: 69) RIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISL HYQKDEEPLFQLKKVRSVNSIMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNP GEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLIS LKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFH VNGGELILIHQNPGEFCVLQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNN SVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYL NVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 338F4 Human single chain OX40L trimer amino acid sequence  without signal sequence  (SEQ ID NO: 70) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVII NCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTT DNTSLDDFHVNGGELILIHQNPGEFCVLALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQ KEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMV ASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 338F5 Human single chain OX40L trimer amino acid sequence   without signal sequence  (SEQ ID NO: 71) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDG FYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASITYKDKVYLNVTTDNTS LDDFHVNGGELILIHQNPGEFCVLSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKV QNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDK VYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 338F6 Human single chain OX40L trimer amino acid sequence  without signal sequence  (SEQ ID NO: 72) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGF YLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSL DDFHVNGGELILIHQNPGEFCVLHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQN NSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVY LNVTTDNTSLDDFHVNGGELILIHQNPGEFCVL 338F5 Human single chain OX40L trimer nucleotide sequence  without signal sequence  (SEQ ID NO: 73) CAGGTCTCTCATAGATACCCACGCATACAATCAATCAAAGTCCAATTTACAGAATATAAG AAGGAAAAAGGATTCATTCTCACATCTCAGAAGGAGGACGAAATCATGAAAGTGCAAAAT AACAGCGTGATAATTAATTGCGACGGTTTTTACCTGATCTCCCTGAAGGGGTATTTCTCC CAGGAGGTCAATATTTCTCTCCACTACCAAAAGGACGAGGAACCTCTCTTCCAGCTCAAG AAAGTTAGAAGCGTCAATTCCCTGATGGTGGCCTCCCTGACTTATAAGGATAAAGTGTAT CTCAATGTTACCACAGATAACACTTCTCTGGATGATTTCCATGTCAATGGTGGAGAGCTC ATCCTCATTCACCAGAACCCTGGGGAGTTCTGCGTACTGTCACACCGGTACCCGCGCATC CAAAGCATAAAAGTTCAATTCACCGAGTATAAAAAAGAGAAGGGTTTCATACTCACATCA CAAAAGGAAGATGAAATTATGAAGGTTCAAAACAACTCTGTTATCATTAACTGCGATGGG TTCTATCTGATTTCACTGAAAGGTTACTTCAGCCAAGAGGTGAACATATCTCTGCATTAT CAGAAAGATGAAGAGCCCCTGTTCCAACTGAAGAAGGTCCGCTCAGTCAACTCACTGATG GTTGCATCCCTCACATATTAAGATAAGGTCTATCTGAATGTGACAACTGACAATACCTCA CTGGATGACTTTCATGTTAACGGAGGCGAACTGATTCTCATACATCAGAATCCAGGAGAG TTCTGTGTCCTCTCCCATCGCTATCCTCGGATTCTTTCTATCAAGGTTCAGTTTACTGAG TACAAAAAAGAAAAGGGATTTATTCTGACCTCTCAAAAAGAGGATGAGATAATGAAGGTC CAGAATAATTCCGTCATTATAAACTGTGACGGCTTCTATCTCATATCCCTCAAGGGGTAC TTTTCACAAGAAGTTAATATATCACTCCATTACCAAAAAGATGAAGAGCCACTCTTTCAA CTGAAAAAAGTCAGATCCGTCAACTCTCTCATGGTCGCTTCTCTCACCTACAAAGACAAA GTTTACCTGAACGTTACTACAGACAACACATCCCTGGACGACTTCCACGTGAATGGCGGG GAACTGATACTGATCCACCAAAATCCCGGCGAATTTTGTGTGCTCTGA OX40L Stalk region variant 1  (SEQ ID NO: 74) ALQVSHRYP OX40L Stalk region variant 2  (SEQ ID NO: 75) SHRYP OX40L Stalk region variant 3  (SEQ ID NO: 76) HRYP Human OX40L extracellular domain amino acid sequence   variant 1 (SEQ ID NO: 77) ALQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGY FSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGG ELILIHQNPGEFCVL Human OX40L extracellular domain amino acid sequence   variant 2 (SEQ ID NO: 78) SHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQE VNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELIL IHQNPGEFCVL Human OX40L extracellular domain amino acid sequence   variant 3 (SEQ ID NO: 79) HRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEV NISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILI HQNPGEFCVL 338F5 Human single chain OX40L trimer-Fc (IgG1) amino acid  sequence with signal sequence underlined  (SEQ ID NO: 80) MEWGYLLEVTSLLAALLLLQRSPIVHAQVSHRYPRIQSIKVQFTEYKKEKGFILTSQKED EIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASL TYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLSHRYPRIQSIKVQFTEYKKE KGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKV RSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLSHRYPRIQS IKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFSQEVNISLHYQK DEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGELILIHQNPGEFC VLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWY VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK AKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSECLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 338F5 Human single chain OX40L trimer-Fc (IgG1) amino acid  sequence without signal sequence  (SEQ ID NO: 81) QVSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDGFYLISLKGYFS QEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTSLDDFHVNGGEL ILIHQNPGEFCVLSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKVQNNSVIINCDG FYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDKVYLNVTTDNTS LDDFHVNGGELILIHQNPGEFCVLSHRYPRIQSIKVQFTEYKKEKGFILTSQKEDEIMKV QNNSVIINCDGFYLISLKGYFSQEVNISLHYQKDEEPLFQLKKVRSVNSLMVASLTYKDK VYLNVTTDNTSLDDFHVNGGELILIHQNPGEFCVLDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSV LTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSPGK Human CD40L (TNFSF5) amino acid sequence  (SEQ ID NO: 82) MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLH EDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSN REASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVN VTDPSQVSHGTGFTSFGLLKL Human CD40L extracellular domain amino acid sequence  (SEQ ID NO: 83) HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETK KENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQ GLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLG GVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human CD40L extracellular domain with stalk fragment 1  (aa 113-261)  (SEQ ID NO: 84) MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer amino acid sequence without signal sequence  (SEQ ID NO: 85) MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer nucleotide sequence without  signal sequence  (SEQ ID NO: 86) ATGCAAAAAGGGGATCAGAATCCTCAAATTGCAGCACATGTCATATCTGAGGCCAGCTCA AAAACAACATCTGTGCTCCAGTGGGCTGAAAAAGGATACTACACCATGAGCAACAACCTC GTTACCCTGGAAAATGGGAAACAGCTGACCGTTAAAAGACAAGGACTCTATTATATCTAT GCCCAAGTCACCTTCTGTTCCAATCGGGAAGCATCTTCACAAGCTCCATTTATCGCCAGC CTCTGCCTCAAGTCCCCCGGTCGGTTCGAGAGAATCCTCCTCAGAGCTGCAAATACCCAC TCTTCCGCCAAACCTTGCGGGCAACAATCCATTCACCTCGGAGGAGTTTTTGAACTGCAA CCAGGGGCTTCTGTGTTTGTCAATGTGACTGATCCAAGCCAAGTGTCTCATGGAACTGGC TTCACTTCCTTTGGCCTCCTCAAACTCATGCAGAAAGGGGACCAAAACCCCCAGATAGCC GCTCACGTTATTTCCGAAGCAAGCTCAAAAACAACATCTGTGCTCCAGTGGGCTGAAAAA GGATACTACACCATGAGCAACAACCTCGTTACCCTGGAGAACGGAAAGCAACTCACTGTG AAGCGGCAGGGGCTGTACTACATATACGCACAAGTGACTTTTTGCAGCAACAGGGAGGCA TCCTCTCAGGCCCCTTTCATTGCCAGCCTCTGCCTGAAGTCCCCCGGTAGATTCGAGAGA ATCCTCCTCAGAGCTGCAAATACCCACTCCTCCGCAAAACCCTGTGGCCAGCAGAGCATC CATCTGGGCGGCGTGTTCGAGCTCCAGCCTGGGGCCTCCGTCTTCGTGTTCGTCACCGAC CCTTCCCAAGTCAGCCACGGCACTGGCTTCACATCCTTTGGCCTCCTCAAACTCATGCAA AAAGGCGATCAGAATCCTCAAATTGCTGCACATGTCATTTCCGAAGCCTCATCCAAAACT ACCTCCGTCCTGCAATGGGCCGAGAAGGGGTATTATACAATGTCAAATAACCTGGTTACT CTGGAAAACGGCAAACAGCTCACTGTTAAGCGCCAAGGTCTCTACTATATATATGCACAA GTTACTTTCTGTTCAAATCGCGAAGCATCATCACAAGCACCATTTATAGCATCACTCTGT CTCAAGTCACCAGGTCGCTTTGAACGCATACTGCTCCGCGCAGCAAATACTCACTCATCA GCAAAACCATGCGGTCAACAATCAATACACCTCGGTGGTGTTTTTGAGCTCCAACCAGGC GCTTCAGTTTTTGTTAATGTTACTGATCCATCACAAGTTTCACATGGTACAGGTTTCACT TCATTTGGTCTGCTCAAACTCTAATAG Human CD40L anchor region and extracellular domain amino acid sequence  (SEQ ID NO: 87) IFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLL NCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAE KGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFE RILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer amino acid sequence with  signal sequence underlined  (SEQ ID NO: 88) MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer-Fc (IgG1) amino acid  sequence with signal sequence underlined (SEQ ID NO: 89) MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHN AKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCECVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human single chain CD40L trimer-Fc (IgG1) amino acid  sequence without signal sequence  (SEQ ID NO: 90) MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMI SRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDW LNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGK Human single chain CD40L trimer-Fc (IgG2) amino acid  sequence with signal sequence underlined (SEQ ID NO: 91) MEWGYLLEVTSLLAALLLLQRSPIVHAMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGY YTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERIL LRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKG DQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVT FCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGAS VFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYT MSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLR AANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLVERKSC VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEV HNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPR EPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Human single chain CD40L trimer-Fc (IgG2) amino acid   sequence without signal sequence  (SEQ ID NO: 92) MQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIY AQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQ PGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEK GYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFER ILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQ KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKLVERKSCVECPPCPAPPVAGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQ DWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPGK Human CD40L Stalk Region  (SEQ ID NO: 93) HRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETK KENSFEMQKGDQ Human CD40L TNF homology domain  (SEQ ID NO: 94) IAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNR EASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNV TDPSQVSHGTGFTSFGLLKL Human CD40L extracellular domain without stalk region amino  acid sequence  (SEQ ID NO: 95) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVF VNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer (2 stalks regions) amino   acid sequence with signal sequence underlined  (SEQ ID NO: 96) MEWGYLLEVTSLLAALLLLQRSPIVHANPQIAAHVISEASSKTTSVLQWAEKGYYTMSNN LVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANT HSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQI AAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNRE ASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVT DPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLV TLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHS SAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human single chain CD40L trimer (2 stalks regions) amino acid sequence without signal sequence  (SEQ ID NO: 97) NPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFC SNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVF VNVTDPSQVSHGTGFTSFGLLKLMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMS NNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAA NTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKLMQKGDQNP QIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSN REASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVN VTDPSQVSHGTGFTSFGLLKL Human CD40L Stalk Region-fragment 1  (SEQ ID NO: 98) MQKGDQ Human CD40L Stalk Region-fragment 2  (SEQ ID NO: 99) FEMQKGDQ Human CD40L Stalk Region-fragment 3  (SEQ ID NO: 100) EMQKGDQ Human CD40L Stalk Region-fragment 4  (SEQ ID NO: 101) QKGDQ Human CD40L Stalk Region-fragment 5  (SEQ ID NO: 102) KGDQ Human CD40L extracellular domain with stalk fragment 2  (aa 111-261)  (SEQ ID NO: 103) FEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYY IYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFE LQPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human CD40L extracellular domain with stalk fragment 3  (aa 112-261)  (SEQ ID NO: 104) EMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYI YAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFEL QPGASVFVNVTDPSQVSHGTGFTSFGLLKL Human CD40L extracellular domain with stalk fragment 4  (aa 114-261)  (SEQ ID NO: 105) QKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYA QVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQP GASVFVNVTDPSQVSHGTGFTSFGLLKL Human CD40L extracellular domain with stalk fragment 5  (aa 115-261)  (SEQ ID NO: 106) KGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQ VTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPG ASVFVNVTDPSQVSHGTGFTSFGLLKL 

1.-213. (canceled)
 214. A polypeptide comprising a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein, wherein at least one of the first, second, or third copies of the extracellular domain or a fragment thereof comprises the stalk region of the TNFSF protein.
 215. The polypeptide of claim 214, wherein the TNFSF protein is selected from the group consisting of glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL), CD40 ligand (CD40L), OX40 ligand (OX40L), 4-1BB ligand, APRIL, BAFF, CD27 ligand, CD30 ligand, EDA, EDA-A1, EDA-A2, Fas ligand, LIGHT, lymphotoxin, lymphotoxin beta, lymphotoxin-alpha, TL1A, TNF-alpha, TRAIL, TRANCE, and TWEAK.
 216. The polypeptide of claim 215, wherein the TNFSF protein is a) GITRL and the polypeptide comprises SEQ ID NO:5 or SEQ ID NO:66; b) OX40L and the polypeptide comprises SEQ ID NO:44, SEQ ID NO: 69, SEQ ID NO:70, SEQ ID NO:71, or SEQ ID NO:72; or c) CD40L and the polypeptide comprises SEQ ID NO:85, or SEQ ID NO:97.
 217. The polypeptide of claim 214, wherein the TNFSF protein is a) GITRL and the stalk region comprises LQLETAK (SEQ ID NO:32); b) OX40L and the stalk region is selected from the group consisting of: QVSHRYP (SEQ ID NO:55), ALQVSHRYP (SEQ ID NO:74), SHRYP (SEQ ID NO:75), and HRYP (SEQ ID NO:76); or c) CD40L and the stalk region is selected from the group consisting of: MQKGDQ (SEQ ID NO:98), FEMQKGDQ (SEQ ID NO:99), EMQKGDQ (SEQ ID NO:100), QKGDQ (SEQ ID NO:101), and KGDQ (SEQ ID NO:102).
 218. The polypeptide of claim 216, which further comprises a human Fc region or an immunoglobulin heavy chain.
 219. The polypeptide of claim 218, wherein the human Fc region is selected from the group consisting of: SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.
 220. The polypeptide of claim 218, which comprises SEQ ID NO:7.
 221. The polypeptide of claim 216, wherein the TNFSF protein is GITR and which activates GITR and/or induces GITR activity.
 222. The polypeptide of claim 214, which: (a) increases cell-mediated immunity; (b) increases T-cell activity; (c) increases cytolytic T-cell (CTL) activity; (d) increases natural killer (NK) cell activity; (e) decreases or inhibits regulatory T-cell (Treg) activity; and/or (f) decreases or inhibits myeloid-derived suppressor cell (MDSC) activity.
 223. A polypeptide encoded by the plasmid deposited with ATCC and assigned designation number PTA-122112.
 224. A bispecific agent comprising: a) the polypeptide of claim 214, and b) an antigen-binding site from an antibody.
 225. The bispecific agent of claim 224, wherein the agent is a homodimer or a heterodimer.
 226. A bispecific agent comprising: a) a first arm comprising a polypeptide comprising a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof, and b) a second arm comprising an antigen-binding site from an antibody.
 227. The bispecific agent of claim 226, wherein the TNFSF protein is GITRL, OX40L, or CD40L.
 228. A pharmaceutical composition comprising the polypeptide of claim 214 and a pharmaceutically acceptable carrier.
 229. A polynucleotide comprising a polynucleotide that encodes the polypeptide of claim
 214. 230. An isolated cell comprising or producing the polypeptide of claim
 214. 231. A method of treating cancer in a subject, a method of inducing, activating, promoting, increasing, enhancing, or prolonging an immune response in a subject, method of inhibiting the growth of a tumor in a subject, method of increasing T-cell activity in a subject, method of increasing cytolytic T-cell (CTL) activity in a subject, method of increasing natural killer (NK) activity in a subject, method of decreasing or inhibiting regulatory T-cell (Treg) activity in a subject, or a method of decreasing or inhibiting myeloid-derived suppressor cell (MDSC) activity in a subject, comprising administering to a subject a therapeutically effective amount of the polypeptide of claim
 214. 232. The method of claim 231, wherein the cancer is selected from the group consisting of colorectal cancer, colon cancer, ovarian cancer, pancreatic cancer, lung cancer, liver cancer, breast cancer, kidney cancer, prostate cancer, gastrointestinal cancer, melanoma, cervical cancer, bladder cancer, glioblastoma, head and neck cancer, lymphoma and leukemia.
 233. The method of claim 231, which further comprises administering at least one additional therapeutic agent.
 234. The method of claim 233, wherein the additional therapeutic agent is a chemotherapeutic agent or an agent that stimulates the immune response.
 235. The method of claim 233, wherein the additional therapeutic agent is an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-CTLA-4 antibody, or an anti-TIGIT antibody.
 236. A method of decreasing or inhibiting regulatory T-cell (Treg) activity or myeloid-derived suppressor cell (MDSC) activity in a subject, or a method of increasing T-cell activity, cytolytic T-cell (CTL) activity, or natural killer (NK) activity in a subject, comprising administering to the subject a therapeutically effective amount of a polypeptide comprising a first, second, and third copy of the extracellular domain of a human tumor necrosis factor receptor ligand superfamily (TNFSF) protein or a fragment thereof capable of binding a receptor of the TNFSF protein.
 237. A method of claim 236, wherein the TNFSF protein is GITRL, OX40L, or CD40L. 